HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 52, 50510-50519, December 27, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/52/50510    most recent M208738200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, X. Articles by Frank, S. J. Search for Related Content PubMed PubMed Citation Articles by Wang, X. Articles by Frank, S. J. Social Bookmarking                      What's this?

Metalloprotease-mediated GH Receptor Proteolysis and GHBP Shedding

DETERMINATION OF EXTRACELLULAR DOMAIN STEM REGION CLEAVAGE SITE^*

Xiangdong Wang, Kai He, Mary Gerhart^§, Yao Huang, Jing Jiang, Raymond J. Paxton^§, Shaohua Yang^¶, Chunxia Lu^¶, Ram K. Menon^¶, Roy A. Black^§, Gerhard Baumann, and Stuart J. Frank^**

From the  Department of Medicine, Division of Endocrinology and Metabolism, and Department of Cell Biology, University of Alabama at Birmingham, Alabama 35294, the ^§ Immunex Corporation, Seattle, Washington 98101, the ^¶ Department of Pediatrics, Children's Hospital of Pittsburgh, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, the  Center for Endocrinology, Metabolism, and Molecular Medicine, Department of Medicine, Northwestern University Medical School and the Veterans Administration Chicago Health System, Lakeside Division, Chicago, Illinois 60611, and the ^** Endocrinology Section, Medical Service, Veterans Affairs Medical Center, Birmingham, Alabama 35233

Received for publication, August 26, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Growth hormone-binding protein (GHBP) is complexed to a substantial fraction of circulating GH. In humans, rabbits, and other species, GHBP derives from proteolytic shedding of the GH receptor (GHR) extracellular domain. In cell culture studies, stimuli such as phorbol ester, platelet-derived growth factor, or serum induce GHR proteolysis, which concomitantly yields shed GHBP in cell supernatants and a cell-associated cytoplasmic domain-containing GHR remnant. This process is sensitive to metalloprotease inhibition, and genetic reconstitution studies identify tumor necrosis factor- converting enzyme (TACE/ADAM-17), a transmembrane metalloprotease, as a GHR sheddase. Stimuli that induce GHR proteolysis render cells less responsive to GH, but the mechanism(s) of this desensitization is not yet understood. In this study, we mapped the rabbit (rb) GHR cleavage site. We adenovirally expressed a C-terminal epitope-tagged rbGHR lacking most of its cytoplasmic domain, purified the remnant protein induced by the phorbol ester, PMA, and derived the cleavage site by N-terminal sequencing of the purified remnant. The N-terminal sequence, ²³⁹FTCEEDFR²⁴⁶, matched perfectly the rbGHR and suggests that cleavage occurs eight residues from the membrane in the proximal extracellular domain stem region. Deletion and alanine substitution mutagenesis indicated that, similar to other TACE substrates, the spacing of residues in this region, more than their identity, influences GHR cleavage susceptibility. Further, we determined that PMA pretreatment desensitized a cleavage-sensitive GHR mutant, but not a cleavage-insensitive mutant, to GH-induced JAK2 activation. These results suggest that inducible GHR proteolysis can regulate GH signaling.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Growth hormone (GH)¹ is an anterior pituitary-derived hormone that exerts somatogenic and metabolic effects by interacting with the GH receptor (GHR) on target cells (1). The GH-bound dimerized GHR causes engagement of intracellular signaling cascades by activating the receptor-associated cytoplasmic tyrosine kinase, JAK2 (2). Activation of pathways including the signal transducer and activator of transcription (STAT) 5, MAP kinase, and phosphatidylinositol 3-kinase pathways, results in expression of insulin-like growth factor-1 and other target genes that contribute to GH effects (3, 4).

A substantial fraction (roughly one-half in humans) of circulating GH is bound to a high-affinity GH-binding protein (GHBP), which corresponds to the extracellular ligand-binding domain of the GHR (5, 6). Depending on species, GHBP is generated by two distinct mechanisms. In rodents, alternative splicing of the GHR mRNA yields a secreted form that includes the extracellular domain (7-9). In humans, rabbits, and other species, GHBP is derived by proteolysis of the GHR (reviewed in Ref. 10). This yields a soluble receptor extracellular domain in a process termed proteolytic GHBP shedding. An obligatory byproduct of proteolytic GHBP shedding is the so-called GHR "remnant", a cytoplasmic domain-containing cell-associated fragment of the receptor that remains after the extracellular domain is shed (11-13).

We have previously shown in cell culture model systems that GHR proteolysis (receptor loss, remnant accumulation, and variable degrees of GHBP shedding) can be induced by several stimuli: 1) pharmacologic activation of protein kinase C by the phorbol ester, PMA; 2) treatment with platelet-derived growth factor (PDGF); or 3) treatment of serum-starved cells with serum (11-14). In each case, the inducible proteolysis is blocked by a metalloprotease inhibitor, and genetic reconstitution studies suggest that tumor necrosis factor- converting enzyme (TACE, also known as ADAM-17, Refs. 15 and 16) can catalyze the processing (14). In cells expressing either murine or rabbit (rb) GHRs, receptor proteolysis is accompanied by a decrease in the ability of subsequent stimulation with GH to elicit JAK2 activation, suggesting that metalloprotease-mediated heterologous desensitization may be a mechanism that impacts cellular responsiveness to GH (12). Conversely, prior treatment with GH, but not with a GH antagonist, renders cells resistant to inducible GHR proteolysis (13). This suggests that GH-induced receptor dimerization or a GH-induced conformational change in the receptor's extracellular dimerization domain and/or stem region may impede access to the cleaving enzyme.

In this study, we examined GHR extracellular domain determinants for metalloprotease-mediated proteolysis. Using adenoviral infection to accomplish abundant receptor expression, we identified a cleavage site in the rbGHR juxtamembrane region of the extracellular domain. We tested the importance of the identity of this site in allowing receptor proteolysis to occur by performing mutagenesis experiments. Finally, by comparing cleavage-sensitive versus cleavage-resistant GHR mutants, we tested the effect of inducible receptor proteolysis on GH-induced signaling.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- PMA and routine reagents were purchased from Sigma Chemical Co. unless otherwise noted. Restriction endonucleases were obtained from New England Biolabs (Beverly, MA). Recombinant hGH was kindly provided by Eli Lilly Co. (Indianapolis, IN). Immunex Compound 3 (IC3), supplied by Immunex Corporation, is identical to Compound 2 (17), except that the napthylalanine side chain is replaced by a tert butyl group. Talon Metal Affinity Resin, used for His-tagged protein purification, was from Clontech.

Cells, Cell Culture, Transfection, and Adenoviral Infection-- HEK-293 cells (a gift from Dr. C. Wu, University of Pittsburgh) were maintained in Dulbecco's modified Eagle's medium (low glucose) (Cellgro, Inc.) supplemented with 7% fetal bovine serum (Biofluids, Rockville, MD) and 50 µg/ml gentamicin sulfate, 100 units/ml penicillin, and 100 µg/ml streptomycin (all Biofluids). Transient transfection was achieved by introducing pCDNA 3.1-driven plasmids encoding GHR mutants (3 µg per transfection; see below for construction) with or without murine JAK2 (1 µg per transfection), as indicated, using LipofectAMINE Plus (Invitrogen) according to the manufacturer's instructions. Adenoviral infection of HEK-293 cells was accomplished using methods previously reported (18).

Plasmid Construction-- The rbGHR cDNA was a kind gift of Dr. W. Wood, Genentech, Inc. Construction of the cDNA encoding rbGHR_{del 297-406} has been described (19, 20). This mutant (referred to herein as "wild-type" or WT) has intact extracellular and transmembrane domains, but lacks residues 297-406 in the cytoplasmic domain (the full-length rbGHR has 620 residues). Thus, the Box 1 region in the proximal cytoplasmic domain is intact, as is the distal two-thirds of the cytoplasmic domain, which contains known GHR tyrosine phosphorylation sites, but the major internalization motif is absent. This GHR mutant retains the ability to respond to GH by allowing tyrosine phosphorylation of itself and JAK2 (see Fig. 7) and STAT5 (data not shown). Ligation of rbGHR_{del 297-406} cDNA into the pcDNA 3.1 () eukaryotic expression vector was accomplished using XbaI and KpnI restriction endonucleases. A C-terminally His₆-tagged version of rbGHR_{del 297-406} (rbGHR_{del 297-406-His}) was prepared by PCR (sequences of oligonucleotide primers used available upon request) and ligated into the pAdlox (as in 18) adenoviral expression shuttle vector using the XbaI and KpnI 5'- and 3'-restriction sites, respectively.

For preparation of cDNA encoding rbGHR_{1-274-Myc-His}, the rbGHR cDNA was used as a template to first amplify the residue 1-274 region (sequences of primers available upon request). This product was ligated into the pcDNA6/Myc-His vector (Invitrogen) using KpnI and EcoRV restriction endonucleases. This yielded a cDNA encoding the first 274 residues of rbGHR followed by a 14-residue spacer and the Myc and His tags on the C terminus (rbGHR_{1-274-Myc-His}) in the pcDNA vector. This receptor mutant lacks most of the cytoplasmic domain, but the presence of the C-terminal Myc tag allows easy detection of the receptor and its remnant with anti-Myc antibodies. To transfer this receptor-encoding cDNA into the pAdEasy adenoviral expression system, the fragment was removed with KpnI and PmeI and ligated into the pAdTrack shuttle vector at the KpnI and EcoRV sites.

cDNAs expression vectors encoding the rbGHR cleavage region mutants, rbGHR-237-239, rbGHR-240-242, rbGHR-242-244, rbGHR-237-239AAA, rbGHR-240-242AAA, and rbGHR-242-244AAA, were each constructed using the ExSite (Stratagene) PCR-based site-directed mutagenesis method and the pCDNA3.1-rbGHR_{del 297-406} as the template. Sequences for the mutagenic oligonucleotides are available upon request. The deletion () mutations removed in-frame the three contiguous amino acids indicated (numbering as in Ref. 5), while the alanine replacement mutations (AAA) changed the indicated residues to alanine. The entire protein coding sequence of each selected mutant cDNA was subjected to dideoxy DNA sequencing (UAB core facility), which verified the presence of the desired mutations and the absence of unwanted mutations.

Generation of Recombinant Adenoviruses-- The methods for generating the adenovirally expressed version of rbGHR_{del 297-406-His} were as referred to in Ref. 18. Briefly, linearized pAdlox-rbGHR_{del 297-406-His} and 5 helper virus DNA were cotransfected into CRE8 cells (21) (an HEK-293 derivative) by LipofectAMINE. The cells were harvested after several days when cytopathic effects became apparent. After lysis by three freeze/thaw cycles, cell debris was pelleted by centrifugation, and supernatant was collected. This supernatant was used for infection of HEK-293 cells. Three further rounds of infection were performed to obtain a high-titer viral stock, which was used for experimental and preparative infection.

To generate adenovirally expressed rbGHR_{1-274-Myc-His}, we used the Adeasy system (22). Briefly, pAdTrack-rbGHR_{1-274-Myc-His} was linearized with PacI and was cotransformed with pAdeasy (helper plasmid) into electrocompetent Escherichia coli BJ5138 cells. Colonies harboring recombinants were selected by virtue of kanamycin resistance. Linearized recombinant plasmid was transfected into HEK-293 cells and high titer viral stock was obtained as above for the pAdlox system.

Antibodies-- The 9E10 anti-Myc monoclonal antibody and the 4G10 monoclonal antiphosphotyrosine (anti-pTyr) antibody were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY), as was the anti-p-JAK2 state-specific antibody reactive with JAK2 that is phosphorylated at residues Tyr¹⁰⁰⁷ and Tyr¹⁰⁰⁸ (reflective of JAK2 activation). The rabbit polyclonal antisera, anti-GHR_cyt-AL47, raised against a bacterially expressed N-terminally His-tagged fusion protein incorporating human GHR residues 271-620 (the entire cytoplasmic domain, Ref. 5), has been previously described (13). Anti-GHR_cyt-mAb is a mouse monoclonal antibody directed against a bacterially expressed GST fusion protein incorporating human GHR residues 271-620 and has been previously described (20). Anti-JAK2_AL33 (directed at residues 746-1129 of murine JAK2) polyclonal serum has been described (23).

Cell Stimulation, Protein Extraction, Immunoprecipitation, Deglycosylation, Electrophoresis, and Immunoblotting-- Serum starvation of HEK-293 transfectants and infectants was accomplished by substitution of 0.5% (w/v) bovine serum albumin (fraction V, Roche Molecular Biochemicals, Indianapolis, IN) for serum in their respective culture media for 16-20 h prior to experiments. Unless otherwise noted, stimulations were performed at 37 °C. Details of the hGH (500 ng/ml) and PMA (at 1 µg/ml) treatment protocols have been described (11-14, 20, 24). Briefly, adherent cells (dish size and number as indicated in figure legends) were stimulated in binding buffer (BB, consisting of 25 mM Tris-HCl (pH 7.4), 120 mM NaCl, 5 mM KCl, 1.2 mM MgCl₂, 0.1% (w/v) bovine serum albumin, and 1 mM dextrose) or Dulbecco's modified Eagle's medium (low glucose) with 0.5% (w/v) bovine serum albumin. Stimulations were terminated by washing the cells once with and then harvesting by scraping in ice-cold phosphate-buffered saline (PBS) in the presence of 0.4 mM sodium orthovanadate (PBS-vanadate). Pelleted cells were collected by brief centrifugation. For each cell type, pelleted cells were solubilized for 15 min at 4 °C in fusion lysis buffer (FLB) (1% (v/v) Triton X-100, 150 mM NaCl, 10% (v/v) glycerol, 50 mM Tris-HCl (pH 8.0), 100 mM NaF, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 10 mM benzamidine, 10 µg/ml aprotinin), as indicated. After centrifugation at 15,000 × g for 15 min at 4 °C, the detergent extracts were electrophoresed under reducing conditions or subjected to immunoprecipitations, as indicated.

For immunoprecipitation of the GHR with the monoclonal anti-GHR_cyt-MAb antibodies, 0.6 µg of purified antibody was used per precipitation. Protein-A Sepharose (Amersham Biosciences) was used to adsorb immune complexes. For deglycosylation of rbGHR mutants, immunoprecipitates were eluted and treatment with endoglycosidase H (Roche Molecular Biochemicals) or vehicle was carried out in accordance with previous published methods (25, 26) and the manufacturer's suggestions. SDS sample buffer eluates were resolved by SDS-PAGE and immunoblotted as indicated.

Resolution of proteins by SDS-PAGE, Western transfer of proteins, and blocking of Hybond-ECL (Amersham Biosciences) with 2% bovine serum albumin were performed as previously described (11-14). Immunoblotting with antibodies 4G10 (1:4000), anti-GHR_cyt-AL47 (1:1000), anti-Myc (1:2000), anti-pJAK2 (1:500), or anti-JAK2_AL33 (1:1000), with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (1:5000) and ECL detection reagents (all from Amersham Biosciences) and stripping and reprobing of blots were accomplished according to the manufacturer's suggestions.

Purification and N-terminal Sequencing of GHR Remnant-- High titered adenovirus stock encoding rbGHR_{1-274-Myc-His}, described above, was used to infect thirty 150 × 25 mm dishes of HEK-293 cells. After 24 h, the medium was removed, and serum-starvation was initiated. After 18 h, the cells were treated with PMA (1 µg/ml, final) for 30 min and then harvested and lysed with FLB, modified to lack EDTA. This detergent cell extract was applied to a TALON Metal Affinity Resin (Co²⁺-TC-Sepharose) column (15 ml). The column was washed twice with 50 ml of wash buffer (PBS with 0.5% (v/v) Triton X-100, and 1 mM phenylmethylsulfonyl fluoride), followed by three washes with 10 mM imidazole in PBS. Proteins bound to the column were eluted with 50 ml of PBS containing 150 mM imidazole. This eluate was concentrated by Centriprep-10K size exclusion. The concentrated sample was precipitated with ice-cold acetone. Proteins in this concentrate were resolved by SDS-PAGE (15% acrylamide) and electrically transferred to a polyvinylidene difluoride membrane (Millipore). A thin strip of the lane containing the purified proteins was cut off and immunoblotted with anti-Myc to verify the location of the band of interest. The remainder of the membrane was stained with Coomassie Brilliant Blue G250 and the Coomassie-stained band corresponding to the specific anti-Myc-identified remnant band was excised from the membrane for N-terminal sequencing by Edman degradation (27) using an AppliedBiosystems 494Ht sequencer.

GHBP Assay-- GHBP activity was measured in conditioned media by a standard GH binding assay, as previously reported (11, 12, 28). Conditioned medium (0.05 or 0.4 ml, as indicated) from cells treated as indicated was incubated with freshly labeled ¹²⁵I-hGH (~0.5 ng) for 45 min at 37 °C. Bound GH was then immediately separated from free GH by gel chromatography on a Sephadex G-100 column at 4 °C. The fraction of GH bound was determined by peak integration. Statistical analysis was performed by analysis of variance followed by Newman-Keuls test, or paired Student's t test as appropriate.

Densitometric Analysis-- Densitometric quantitation of ECL immunoblots was performed using a video camera and the Image 1.49 program (developed by W. S. Rasband, Research Services Branch, NIMH, Bethesda, MD). For normalization of GHBP shedding in the experiments in Fig. 5B, the relative abundance of transfected GHRs among transfections within an each experiment was estimated by densitometric scanning of the mature GHR form present in the immunoblot (such as in Fig. 5A). The measured GHBP shed into the supernatant of each sample was thus corrected by the abundance of receptor expressed within that transfection to facilitate comparison between wild-type and mutants. In the experiments shown in Fig. 8, the relative degree of basal and GH-induced specific JAK2 tyrosine phosphorylation in cell extracts from cells pretreated with PMA versus Me₂SO vehicle was estimated by measuring the intensity of the pJAK2 signal, normalized for the abundance of JAK2 in the sample (by anti-JAK2_AL33 immunoblot signal) for each condition. JAK2 activation induced by GH in the Me₂SO-pretreated samples within each transfection was considered 100%. As indicated when graphically shown, pooled data from several experiments are displayed as the mean ± S.E. The significance of differences of pooled results is estimated by unpaired Student's t tests.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Adenovirally Expressed C-terminal Epitope-tagged rbGHR Cytoplasmic Domain Mutants Undergo Inducible Proteolytic GHBP Shedding in HEK-293 Cells-- To better characterize GHR structural determinants for proteolytic shedding and map the cleavage site, we sought to develop a transient expression system that would ensure high GHR levels, but maintain the characteristics of the system we previously defined. In prior work, we characterized a rbGHR mutant that has an in-frame deletion of residues 297-406 in the cytoplasmic domain. rbGHR_{del 297-406}, which lacks the internalization and UbE motif (29, 30), is highly expressed at the cell surface, binds GH normally, couples to GH-induced JAK2 activation, and, when stably expressed in CHO cells, undergoes inducible metalloprotease-mediated, GH-inhibitable proteolysis, and GHBP shedding (13, 19). Infection of cells with replication-defective adenoviral vectors is an increasingly common method of achieving high-level eukaryotic protein expression. We first tested whether rbGHR_{del 297-406} could be expressed adenovirally in human HEK-293 cells and, if so, would behave similarly regarding proteolytic shedding in this system as in those we previously studied.

We used the pAdlox system to prepare infectious adenoviral particles encoding a C-terminally His-tagged rbGHR_{del 297-406}, as described under "Experimental Procedures." Infection of HEK-293 cells was efficient, with greater than 80% of cells exhibiting infection when GFP signal was detected by microscopy (data not shown). As seen in Fig. 1, immunoblotting with anti-GHR_cyt-AL47, which recognizes epitopes in the GHR cytoplasmic domain (13), detected the receptor in unfractionated detergent extracts of the infected HEK-293 cells (lane 1). As we previously observed (13, 19), this receptor mutant, like the wild-type rbGHR, appears as a mixture of indistinct and sharp bands, which collectively migrate at roughly 75-100 kDa, consistent with its glycosylation (more below). A more rapidly migrating and more distinct form was also detected by anti-GHR_cyt-AL47 at roughly 43 kDa (arrow). The abundance of this form was increased acutely in response to treatment with PMA (lane 2 versus lane 1), consistent with it being the GHR remnant we have previously described (13). We tested the effect of inclusion of the hydroxamate-based metalloprotease inhibitor, IC3, on this process. IC3 prevented the PMA-induced appearance of the remnant (lane 3 versus lane 2), but did not substantially change the basal amount of remnant (lane 4 versus lane 1). In contrast, when IC3 was added in the absence of PMA for 20 h in the serum starvation medium, the abundance of the remnant present basally (presumably reflecting basal proteolysis during the serum starvation period) was greatly diminished (lane 6 versus lane 5). These findings strongly suggest that both basal and inducible receptor proteolysis were occurring in this adenoviral system in a fashion qualitatively similar to our previous observations in other systems (11-13). This was further supported by our ability to measure PMA-induced shedding of ample GHBP into the medium of these cells and inhibition of this shedding by IC3 (Fig. 1B).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Inducible metalloprotease-mediated receptor proteolysis and GHBP shedding of adenovirally expressed rbGHRdel 297-406. A, receptor proteolysis. HEK-293 cells were adenovirally infected with pAdlox-rbGHRdel 297-406-His as described under "Experimental Procedures." Serum-starved cells (one 90% confluent well of a 6-well plate per sample) were exposed in samples 1-4 to PMA (+) or the Me2SO vehicle () for 45 min in the presence (+) or absence (, Me2SO vehicle) of IC3 (50 µM). For samples 5 and 6, cells were serum-starved for 20 h in the presence (+) or absence (, Me2SO vehicle) of IC3. At the end of the incubations, one-sixth of the detergent cell extracts were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. The positions of the GHR and the remnant are indicated, as are the migration of prestained molecular mass markers. The data shown are representative of three such experiments. B, GHBP shedding. Supernatants from samples such as in A were harvested prior to cell lysis. GHBP content was measured in the supernatants, as described under "Experimental Procedures," and is graphically expressed as percent of radiolabeled GH bound per 50 µl of medium. The data are from n = 2 and are graphed as the mean ± S.E. Note that IC3 inhibits both PMA-induced (lanes 1-4) and constitutive (lanes 5 and 6) GHR proteolysis and GHBP shedding for rbGHRdel-297-406 expressed in this system (all differences were significant at p < 0.01, except for the comparison of IC3 treatment alone with PMA plus IC3 (lane 3 versus 4)).

It is known that a naturally occurring GHR splice variant lacking nearly the entire cytoplasmic domain generates substantial amounts of proteolytically shed GHBP (31, 32). For remnant purification, we designed and adenovirally expressed a rbGHR mutant, rbGHR_{1-274-Myc-His}, that encodes the first 274 residues of the receptor followed by C-terminal Myc and His tags. Thus, like the naturally occurring variant, the rbGHR extracellular and transmembrane domains and only the first four cytoplasmic domain residues are present in this mutant. After infection of HEK-293 cells, the glycosylated rbGHR_{1-274-Myc-His} was detected by anti-Myc immunoblotting of cell extracts at the expected migration in SDS-PAGE (Fig. 2A, lane 1, bracket). Just as seen for rbGHR_{del 297-406}, a rbGHR_{1-274-Myc-His} remnant of the expected M_r was detected basally and increased in abundance by PMA treatment (Fig. 2A, lanes 1 and 2, arrow). Further, remnant abundance was substantially lessened in the presence of IC3 (Fig. 2A, lane 3 versus lane 2) and PMA-induced, IC3-inhibitable GHBP shedding into the cell supernatant was also readily detected (Fig. 2B). In addition, pretreatment of the cells with GH prior to acute PMA exposure blunted the PMA-induced appearance of the remnant (Fig. 1C), indicating that this property of the proteolytic shedding system that we have previously documented (13) was also intact in this system.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Inducible metalloprotease-mediated receptor proteolysis and GHBP shedding of adenovirally expressed rbGHR1-274-Myc-His and its inhibition by GH. A, receptor proteolysis. HEK-293 cells were adenovirally infected with pAdeasy-rbGHR1-274-Myc-His as described under "Experimental Procedures." Serum-starved cells were treated with PMA, vehicle, or IC3, as in Fig. 1. Detergent cell extracts were resolved by SDS-PAGE and immunoblotted with anti-Myc. The positions of the GHR, its remnant, and the prestained molecular mass markers are shown. The data shown are representative of three such experiments. B, GHBP shedding. GHBP shed into the cell supernatants from experiments such as in A was measured as in Fig. 1. The data are from n = 2 and are graphed as the mean ± S.E. Differences among the means are all statistically significant (p < 0.05), except for the comparison of IC3 treatment alone with PMA plus IC3. Note that IC3 inhibits PMA-induced and constitutive GHR proteolysis and GHBP shedding for adenovirally expressed rbGHR1-274-Myc-His. C, effect of GH on receptor proteolysis. Cells infected as in A and B were serum-starved overnight in the presence (+) or absence () of GH (500 ng/ml) and then treated for 45 min with (+) or without (, Me2SO vehicle) PMA. Detergent cell extracts were resolved by SDS-PAGE and immunoblotted with anti-Myc. Only the portion of the blot including the remnant is shown. (Receptor abundance was not changed by GH incubation (not shown)). The data shown are representative of two such experiments. Note that GH inhibits PMA-induced proteolysis in this system.

N-terminal Sequencing of the Purified GHR Remnant-- These results suggested a strategy for identification of the inducible, metalloprotease-mediated GHR cleavage site utilizing adenoviral infection to express rbGHR_{1-274-Myc-His} in HEK-293 cells in order to purify the remnant via the C-terminal His tag. The scaled-up purification strategy is outlined in Fig. 3A. Thirty 150-mm² dishes of HEK-293 cells were infected with adenovirus encoding rbGHR_{1-274-Myc-His} and, after serum starvation, were treated with PMA for 30 min at 37 °C. The cells were harvested and solubilized in a Triton X-100-based lysis buffer. Extract was subjected to metal affinity chromatography and the imidazole eluate was concentrated and resolved by SDS-PAGE. After western transfer, we compared the patterns of anti-Myc immunoblotting (of a small fraction of the eluate) and Coomassie staining (of over 90% of the eluate) in the relevant region of the gel (not shown) and thereby identified the putative purified remnant in the Coomassie-stained material. This band was excised and the protein was eluted and subjected to N-terminal sequencing by Edman degradation. As seen in Fig. 3B, the resulting amino acid sequence was FTCEEDFR, matching perfectly the known rbGHR extracellular domain sequence ²³⁹FTCEEDFR²⁴⁶. This clearly confirmed that we indeed purified the intended protein and strongly suggested that the cleavage occurs between Pro²³⁸ and Phe²³⁹ in the extracellular juxtamembrane region of the receptor, with Phe²³⁹ eight residues N-terminal to the first transmembrane domain residue (5). Of note, this is a region of highly conserved amino acid sequence between the rabbit and human GHRs (Fig. 3B). It is also a region of the GHR extracellular domain about which little is known of the structure; the bacterially expressed hGHR extracellular domain previously studied crystallographically encoded only residues 1-238 (33).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Purification of GHR remnant and sequencing of its N terminus. A, purification strategy and sequence data. The strategy for large-scale adenoviral expression of rbGHR1-274-Myc-His and the PMA-induced generation of and purification of its remnant protein, as described under "Experimental Procedures" and the text, is illustrated. Edman degradation sequencing yielded the sequence FTCEEDFR as the N-terminal sequence of the purified remnant (roughly 3.1 pmol was recovered, based on the sequencing results). B, comparison of rabbit (rb) and human (h) GHR amino acid sequence in the juxtamembraneous extracellular (ECD), transmembrane (TMD, boxed), and juxtamembraneous intracellular (ICD) domain regions.

Proteolytic Susceptibility of rbGHR Cleavage Region Mutants-- There is no clear consensus sequence for cleavage among the various TACE substrates (34). While the determinants for proteolysis are not known in all cases, the distance of the site of cleavage from the membrane or the position of the cleavage site relative to globular extracellular domain regions have been suggested as important determinants (35). Given our remnant N-terminal sequencing results, we sought to determine the degree to which the receptor susceptibility to proteolysis depended on the intactness of the site we identified. In these experiments, we used the rbGHR_{del 297-406} characterized in Fig. 1 in HEK-293 cells and in our previous work in other cells (13, 19, 20). For simplicity, this is referred to as the "wild-type" (WT) receptor for the remainder of this study because it contains the normal rbGHR sequence in the extracellular and transmembrane domains. In the framework of this receptor, we prepared cDNAs encoding mutations in the extracellular domain region encompassing and near to the cleavage site. (These mutants are referred to by the nature of the extracellular domain mutations, without reference to the in-frame cytoplasmic domain deletion that they all share.) The mutants (diagrammed in Fig. 4) were designed to test whether the identity of the residues in the extracellular domain stem region versus the length of this region was most critical for susceptibility to receptor proteolysis. Three stem region in-frame internally deleted mutants were engineered that each lacked the three contiguous residues (rbGHR-237-239, rbGHR-240-242, and rbGHR-242-244). Three-residue deletions were chosen because they would be predicted to be least disruptive of a helical structure, if this region indeed adopts such a structure. For comparison, mutants were engineered that in each case replaced the three residues with alanine, thus maintaining the number of amino acids present, but changing the identity of those residues (rbGHR-237-239AAA, rbGHR-240-242AAA, and rbGHR-242-244AAA). All of the mutant cDNAs and the WT receptor cDNA were in the context of the pCDNA 3.1 eukaryotic expression vector plasmid.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   GHR mutants to be studied. rbGHRdel 297-406, which has an in-frame internal deletion of 110 cytoplasmic domain residues, including the internalization and UbE motifs (see text) is diagrammed. This is referred to as wild-type (WT), for comparison to the other mutants, which also have deletions or alanine substitutions in the juxtamembraneous extracellular domain. For each deletion () mutation, the residues deleted are indicated by their absence and replacement with an underline. For mutants in which contiguous groups of three residues are replaced by alanine (AAA), the residue changes are indicated. The position of phenylalanine-239 (Phe239) and the mapped (putative) cleavage site (bold arrowhead) between residues 238 and 239 are indicated.

HEK-293 cells were transfected with the WT rbGHR plasmid versus the empty vector control and the serum-starved cells were treated with PMA or its vehicle for 45 min prior to detergent extraction and resolution by SDS-PAGE. As expected, immunoblotting with anti-GHR_cyt-AL47 specifically revealed the GHR and remnant proteins (Fig. 5A, lanes 3 and 4 versus 1 and 2), consistent with the results of the adenoviral expression experiments in Fig. 1. Each of the receptor mutants was similarly expressed by transfection in comparison to the WT (Fig. 5A, lanes 5-16). In each case, the GHR was specifically detected by anti-GHR_cyt-AL47 immunoblotting in a pattern indistinguishable from the WT receptor. Notably, however, both the basal and PMA-induced remnant were absent for rbGHR-237-239 (lanes 5 and 6) and remnant, though detectable, was dramatically reduced in abundance for rbGHR-240-242 and rbGHR-242-244 (lanes 9 and 10 and 13 and 14). These results suggest that removal of three amino acids in these regions substantially inhibited proteolytic remnant generation. In contrast, basal and PMA-induced remnant accumulation appeared normal for rbGHR-237-239AAA and rbGHR-240-242AAA (lanes 7 and 8 and 11 and 12). For rbGHR-242-244AAA, remnant was also easily detected, but a more complicated pattern of remnant bands (an apparent doublet) was observed in comparison to the WT or other AAA mutant receptors (lanes 15 and 16). It is not clear if this finding reflects utilization of a cleavage site(s) in rbGHR-242-244AAA that differs from that of the others.

View larger version (38K): [in this window] [in a new window]   Fig. 5.   Proteolysis and shedding of the GHR mutants. A, receptor proteolysis. HEK-293 cells were transiently transfected with either vector (pcDNA 3.1) only (lanes 1 and 2), or pcDNA 3.1-driven WT (rbGHRdel 297-406) or mutants, as indicated (lanes 3-16), as described under "Experimental Procedures." Serum-starved cells (one 90% confluent well of a 6-well plate per sample) were exposed to PMA (+) or the Me2SO vehicle () for 45 min. Detergent cell extracts were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. The positions of the GHR, its remnant, and the prestained molecular mass markers are shown. The data shown are representative of three such experiments. Note that the degree of basal (PMA-independent) proteolysis observed varied somewhat between transfections for those mutants that exhibited PMA-inducible proteolysis. B, GHBP shedding. Supernatants (0.4 ml) from samples such as in A were harvested prior to cell lysis. GHBP content was measured in the supernatants, as described under "Experimental Procedures." Within each of three independent experiments, the amount of shed GHBP was normalized for the abundance of the transfected receptor by densitometry. Data are graphically expressed for each mutant as the normalized GHBP shed relative to that shed by WT within each experiment and are plotted as mean ± S.E. (n = 3). PMA induced statistically significant (p < 0.05) GHBP shedding over basal for WT, rbGHR-237-239AAA, and rbGHR-240-242AAA and nearly statistically significant (p < 0.1) GHBP shedding for rbGHR-242-244AAA. For rbGHR-242-244AAA, though shedding was observed, the GHBP amount shed was significantly (p < 0.04) less than that shed by WT.

In companion experiments, supernatants were taken from each sample after the 45-min exposure to PMA or vehicle and assayed for the presence of GHBP (Fig. 5B). GHBP was inducibly shed into the supernatant in response to PMA from cells expressing WT, rbGHR-237-239AAA, rbGHR-240-242AAA, and, to a lesser extent, rbGHR-242-244AAA, but no GHBP was shed basally or inducibly from cells expressing rbGHR-237-239. Lower level, non-inducible, or minimally inducible shedding was observed from cells expressing rbGHR-240-242 and rbGHR-242-244, respectively. Of interest, rbGHR-237-239AAA and especially rbGHR-240-242AAA appears even more efficient in GHBP shedding than the WT. Constitutive GHBP shedding among these mutants paralleled the patterns seen with PMA induction, suggesting that PMA-induced and basal shedding share common mechanisms. These GHBP-shedding results are consistent with the pattern of GHR proteolysis observed by anti-GHR immunoblotting for these mutants. The findings in Fig. 5 suggest that the exact identity of the residues present at or near the GHR cleavage site may not be as critical as the preservation of the length of the stem region in allowing basal and inducible receptor proteolysis and GHBP shedding. However, the incomplete recovery of normal cleavage and shedding the 242-244AAA mutant as compared with rbGHR-242-244 suggests that the identity of these more membrane-proximal residues may also be of importance.

Processing and Signaling Properties of GHR Cleavage Region Mutants-- The GHR is a glycoprotein that undergoes processing characteristic of surface glycoprotein receptors after synthesis in the endoplasmic reticulum in transit through the Golgi apparatus to the cell surface. Although it is uncertain where in the cell the metalloprotease-mediated GHR proteolysis and GHBP shedding occurs, several studies suggest that the receptor pool that includes mature (terminally glycosylated) GHRs is a target for proteolysis (12, 36, 37). Given their resistance to proteolysis, we considered the possibility that rbGHR-237-239, rbGHR-240-242, and rbGHR-242-244 could be mutated in such a way as to be inappropriately routed within the cell, rendering them inaccessible to the cleaving apparatus. To examine this, we compared the glycosylation patterns of the mutants with that of the WT by testing the sensitivity of each to digestion with endoglycosidase H (endo H) (Fig. 6). Sensitivity to deglycosylation by endo H suggests the presence of immature high-mannose forms of a glycoprotein, which reside at a pre-trans-Golgi intracellular location rather than at the cell surface; in contrast, the acquisition of endo H resistance is characteristic of a glycoprotein with a mature pattern of carbohydrate chains that are added at a post-trans-Golgi location.

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Endoglycosidase H sensitivity of WT and GHR mutants. HEK-293 cells were transiently transfected with either pcDNA 3.1-driven WT (rbGHRdel 297-406) or rbGHR-237-239 or rbGHR-237-239AAA mutants, as indicated. Serum-starved cells (one 90% confluent 60 × 15 mm dish per sample) were harvested and detergent-extracted. GHRs were immunoprecipitated with anti-GHRcyt-mAb. Precipitated proteins were divided equally and treated either with (+) or without () endoglycosidase H and eluates were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. The positions of the endo H-resistant and endo H-sensitive forms of each receptor are indicated, as is the position of the deglycosylated form that appears in response to endo H (endo H-sensitive degly). Note that both mutants displayed the same pattern of endo H sensitivity as did the WT. The data shown are representative of two such experiments.

WT and mutant rbGHRs were transiently expressed in HEK-293 cells and immunoprecipitated from cell extracts with a monoclonal antibody directed at the GHR cytoplasmic domain (anti-GHR_cyt-Mab) (20). Immunoprecipitates were treated with endo H or its vehicle control and eluates were resolved by SDS-PAGE and immunoblotted with anti-GHR_cyt-AL47. As in unfractionated cell extracts in Figs. 1 and 5, the immunoprecipitated intact WT receptor was detected in two forms, a broad set of bands migrating at roughly 85-100 kDa and a doublet at roughly 75 kDa (Fig. 6, lane 1). As we and others (26, 38, 39) have previously observed for rbGHR in other cells and tissues, endo H treatment caused the sharper bands to migrate more rapidly, but did not affect the migration of the broad 85-100-kDa receptor form (lane 2 versus 1). This pattern is highly consistent with that seen for the full-length rbGHR and indicates that the endo H-resistant broad set of bands constitutes the mature receptor and the sharper, endo H-sensitive bands are the immature (or precursor) forms. As seen in Fig. 6, lanes 3-6, the pattern of endo H susceptibility for rbGHR-237-239 and rbGHR-237-239AAA mimicked that of the WT receptor, strongly suggesting that each exhibited unimpaired processing in the protein secretory pathway, despite the presence of alterations in the membrane-proximal extracellular domain. Identical results were obtained for each of the other deletants (rbGHR-240-242 and rbGHR-242-244) and alanine substitution mutants (rbGHR-240-242AAA and rbGHR-242-244AAA) (data not shown). It is thus unlikely that improper routing of the deletion mutants accounts for their resistance to cleavage. Further, the WT and each of the mutant GHRs were detected by immunoprecipitation with our monoclonal antibody, anti-GHR_ext, which is a conformationally sensitive antibody directed at the extracellular domain (20), thus suggesting that the mutants do not differ from the WT in this respect either (data not shown).

To further verify the integrity of the receptor mutants, we assessed their abilities to initiate signaling in response to GH (Fig. 7). HEK-293 cells were cotransfected with expression plasmids encoding either WT or mutant GHRs (or empty vector as a negative control) and murine JAK2. Serum-starved cells were treated with GH or vehicle for 10 min and detergent cell extracts were resolved by SDS-PAGE. Immunoblotting with a state-specific antibody that recognizes only the activated form of JAK2 (anti-p-JAK2) revealed GH-induced JAK2 tyrosine phosphorylation in cells expressing the WT or each of the mutant receptors (Fig. 7A, upper panel, lanes 3-16), but not in cells expressing JAK2 alone (lanes 1 and 2). Reprobing with anti-JAK2_AL33 (Fig. 7A, lower panel) verified the presence of the JAK2 in each lane. In a separate immunoblot, the resolved proteins were probed with anti-pTyr (Fig. 7B). By comparing the migration of the bands detected with this antibody versus those detected by anti-JAK2_AL33 and anti-GHR_cyt-AL47 (not shown), we observed GH-induced tyrosine phosphorylation of the GHR (only the mature form) and JAK2 again in cells expressing both WT and mutant receptors, but not in those expressing JAK2 alone. We note that though GH-inducible activation of tyrosine phosphorylation was observed for all of the mutants, substantial basal activation was also apparent, in particular for the mutants with changes (either deletion or alanine substitution) in the 242-244 region. We do not yet know the basis for this finding. Collectively, however, the results in Fig. 7 suggest that the WT and each of the mutant receptors were present at the cell surface, capable of binding GH, and responsive to GH stimulation.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   WT and mutants are each able to respond to GH with JAK2 and GHR tyrosine phosphorylation. HEK-293 cells were transiently transfected with either vector (pcDNA 3.1) plus pcDNA 3.1 JAK2 (lanes 1 and 2), or pcDNA 3.1-driven WT (rbGHRdel 297-406) or mutants, as indicated, plus pcDNA 3.1 JAK2, (lanes 3-16). Serum-starved cells (one 90% confluent 60 × 15 mm dish per sample) were treated with (+) or without () GH for 10 min. Detergent cell extracts were resolved by SDS-PAGE and sequentially immunoblotted with anti-pJAK2 (A, upper panel), anti-JAK2AL33 (A, lower panel), and anti-pTyr (B). The positions of tyrosine-phosphorylated JAK2, total JAK2, and tyrosine-phosphorylated GHR are indicated. The data shown are representative of three such experiments.

GHR Proteolysis and Desensitization of Signaling-- In previous work, we demonstrated that treatment of serum-starved cells with PMA, PDGF, or serum-rendered cells less responsive to GH for JAK2 activation and that this desensitization was prevented by the metalloprotease inhibitor, IC3 (12). We hypothesized that the heterologous desensitization of GH signaling by these agents was related to their abilities to cause metalloprotease-mediated GHR proteolysis. Others have recently suggested that activation of PKC isoforms in myeloid progenitor cells may primarily cause decreased interleukin-3 (IL-3)-induced JAK2 activation by serine/threonine phosphorylation of JAK2 rather than by proteolytic effects on the IL-3 receptor, a cytokine receptor family member (40). We sought to use a cleavage-resistant GHR mutant to test further the underlying mechanism of PMA-induced desensitization of GH signaling in our system. As seen in Fig. 8A, expression of the WT receptor with JAK2 in HEK-293 cells allowed GH-induced activation of JAK2 (lanes 1 and 2), as was also observed in Fig. 7. As we have seen in other cell systems, pretreatment with PMA substantially decreased the activation induced by GH in this transient reconstitution system (Fig. 8A, lane 4 versus lane 2).

View larger version (19K): [in this window] [in a new window]   Fig. 8.   GHR proteolysis and heterologous desensitization of signaling. A, wild type. HEK-293 cells were transiently transfected with pcDNA 3.1-WT (rbGHRdel 297-406) plus pcDNA 3.1 JAK2 and divided into four equivalent samples. Serum-starved cells (one 90% confluent 60 × 15 mm dish per sample) were exposed to PMA (+) or the Me2SO vehicle () for 30 min prior to treatment with (+) or without () GH (500 ng/ml) for 10 min. Detergent cell extracts were resolved by SDS-PAGE and sequentially immunoblotted with anti-pJAK2 (upper panel) and anti-JAK2AL33 (lower panel). The positions of tyrosine-phosphorylated JAK2 and total JAK2 are indicated. Note the decreased GH-induced JAK2 activation in the sample pretreated with PMA. The data shown are representative of three such experiments. B, non-cleavable (rbGHR-237-239) and cleavable (rbGHR-237-239AAA) mutants. HEK-293 cells were transiently transfected with pcDNA 3.1- rbGHR-237-239 or pcDNA 3.1-rbGHR-237-239AAA plus pcDNA 3.1 JAK2 and each cotransfected pool was divided into four equivalent samples. Serum-starved cells (one 90% confluent 60 × 15-mm dish per sample) were exposed to GH with or without PMA pretreatment, as in A. Detergent cell extracts were resolved by SDS-PAGE in duplicate and immunoblotted with anti-pJAK2 (upper panel) and anti-JAK2AL33 (lower panel). The positions of tyrosine-phosphorylated JAK2 and total JAK2 are indicated. In separate experiments (not shown), the specific 125I-hGH binding capacities of WT, rbGHR-237-239, and rbGHR-237-239AAA transiently expressed in HEK-293 cells were quite similar (varying less than 10% between them). There was no significant 125I-hGH binding for HEK-293 cells transfected with the vector only. C, densitometric analysis of B. Multiple experiments for each receptor mutant shown in B (rbGHR-237-239, n = 3; rbGHR-237-239AAA, n = 3) were analyzed densitometrically. Relative specific JAK2 tyrosine phosphorylation (mean ± S.E.), determined as under "Experimental Procedures," is plotted. For each mutant, the level induced by GH in the absence of PMA pretreatment is considered 100%.

We next compared the cleavage-resistant mutant, rbGHR-237-239, with the alanine-replaced, cleavage-sensitive rbGHR-237-239AAA in this paradigm. As seen for WT GHR, pretreatment of cells expressing rbGHR-237-239AAA with PMA resulted in decreased GH-induced JAK2 activation, as assessed by anti-p-JAK2 immunoblotting (Fig. 8B, upper panel, lane 8 versus lane 6). Reprobing of this blot with anti-JAK2 showed that this decreased activation was not accounted for by a decrease in JAK2 abundance (lower panel, lanes 5-8). In contrast, PMA pretreatment of cells expressing rbGHR-237-239 did not render them resistant to GH-induced JAK2 activation (Fig. 8B, upper panel, lane 4 versus lane 2). Fig. 8C displays graphically the pooled results of three such experiments. These data strongly suggest that the susceptibility of the GHR to inducible proteolytic shedding is related to its capacity for desensitization in response to acute treatment with PMA.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Metalloprotease-mediated proteolysis of the transmembrane GHR is likely to subserve several possible outcomes relevant for GH action. In those species that use this activity as a major mechanism of GHBP generation, the presence of the GHBP may affect GH action either locally or at a distant location, though this aspect of GHBP action is as yet incompletely studied. Further, we have shown that in cells expressing either the endogenous GHR or a heterologous rbGHR, metalloprotease-mediated GHR proteolysis is detectable and can regulate cellular sensitivity to GH stimulation (Ref. 12 and this study).

The current study extends our knowledge of the mechanisms and roles of regulated GHR proteolysis in several important ways. We used an adenoviral infection approach in HEK-293 cells to generate high levels of truncated GHR protein, allowing purification of GHR remnant and analysis by N-terminal sequencing of the cleavage site that resulted in the remnant. Although there are potential pitfalls with this approach, features of our analysis enhance our confidence that our results are relevant for understanding GHR proteolysis as it occurs in cells. In our initial experiments, we showed that both rbGHR_{del 297-406} (which was used later as the backbone for mutagenesis) and rbGHR_{1-274-Myc-His} (which was used for the purification) behaved in this system as we had previously observed for expression of full-length receptors and rbGHR_{del 297-406} in other cell types (11-13). That is, despite infectious overexpression: 1) rbGHR_{del 297-406} and rbGHR_{1-274-Myc-His} underwent basal and PMA-induced cleavage and GHBP shedding with kinetics similar to what we previously showed; 2) PMA-induced proteolysis and GHBP shedding was inhibited by IC3; and 3) GH antagonized PMA's ability to cause receptor cleavage. In addition, rbGHR_{1-274-Myc-His} is analogous to a naturally occurring alternatively spliced GHR form that is known to effectively yield proteolytically shed GHBP in other systems (31, 32).

Our determination of the GHR cleavage site is the first such reported using these methods, to our knowledge. Phe²³⁹, the N-terminal residue present in the purified rabbit remnant, is eight residues N-terminal to the first predicted transmembrane amino acid (Phe²⁴⁷) of the receptor. This is a region of the rbGHR that has very strong similarity to the human GHR (23 of 25 residues in the proximal extracellular domain, including Phe²³⁹, are identical; see Fig. 3B). It is notable that to enable high level bacterial expression for crystallographic studies of the human GHBP, deVos et al. (33) expressed a recombinant hGHR_1-238 molecule. The cleavage site that would yield such a GHBP is between Q²³⁸ and F²³⁹ of the hGHR, exactly analogous to the rbGHR cleavage site we have empirically determined in this study. We recognize, however, that our methods may not identify all cleavage sites used in intact animals and humans. Incomplete attempts to sequence the C terminus of the GHBP purified from rabbit serum by Leung et al., (5) for example, suggested that Phe²³⁹ may be included. Thus, we consider it possible that another secondary cleavage site(s) may also be utilized to some degree in GHBP generation.

Using our cleavage site sequence information, we studied the effects on rbGHR proteolysis and inducible GHBP shedding of introducing small internal deletion or alanine replacement mutations in and around the observed cleavage site. The most dramatic effect was seen with the rbGHR-237-239 mutant, in which three residues inclusive of the cleavage site were deleted. This mutant failed to undergo proteolysis or GHBP shedding. Yet, its glycosylation pattern suggested that it was routed normally in the biosynthetic pathway and it retained the ability to mediate JAK2 activation and undergo tyrosine phosphorylation in response to GH. In distinction, replacement of these three deleted amino acids with alanine residues (rbGHR-237-239-AAA) restored normal (or even supranormal) proteolysis and inducible GHBP shedding. The findings with these two mutants strongly suggest that the presence of amino acids in this region, rather than the identity of those amino acids, is critical for receptor cleavage. Similar findings emerged from analysis of two other internal deletants, rbGHR-240-242 and rbGHR-242-244, in comparison to the AAA replacement versions of each (rbGHR-240-242-AAA and rbGHR-242-244-AAA). However, the inhibition of cleavage and shedding was not as complete with either of these two deletants as it was for rbGHR-237-239. Furthermore, while the 240-242AAA mutant exhibited a substantially greater propensity to cleavage than the WT receptor, the 242-244AAA mutant was more resistant to cleavage. Collectively, these data suggest that the rbGHR cleaving machinery appears to recognize features in the proximal extracellular domain that relate to the length of the stem region and/or the distance from the plasma membrane to the receptor's first globular extracellular subdomain. Apparently, proper access for cleavage is not allowed if this distance is too small.

This conclusion is supported by the recent work of Conte et al. (41), who examined by mutagenesis the influence of the hGHR extracellular juxtamembrane region on inducible GHBP shedding. No cleavage site was mapped in that study, but deletion of residues 242-244 was seen as particularly deleterious to receptor shedding. Our rbGHR-242-244 was also relatively deficient in inducible (or basal) shedding. However, because our analysis also included a biochemical assessment of proteolysis (by detection of remnant), we observed that both rbGHR-242-244 and rbGHR-242-244AAA generated a remnant pattern that differs slightly from that of the wild-type, rbGHR-237-239-AAA, or rbGHR-240-242-AAA (see Fig. 5A). Thus, in addition to quantitatively lessening cleavage, mutation of residues 242-244 may also change the preferred cleavage site utilized. We also note that mutation of the 242-244 region, while it did not abrogate JAK2 activation and receptor tyrosine phosphorylation, appeared to lessen the GH-inducible component of activation in our system when compared with the WT or the other mutants. This suggests that this region may also be critical for aspects of GHR function other than regulation of proteolysis. This issue is worthy of further investigation.

We previously demonstrated that fibroblasts from a mouse with a homozygous disruption of the gene encoding tumor necrosis factor- converting enzyme (TACE/ADAM-17) failed to shed GHBP in response to PMA treatment and that TACE expression in these cells rescued inducible shedding (14). This implicated TACE, a transmembrane member of the ADAM (a disintegrin and metalloprotease) subfamily of the adamalysin family of metalloproteases (15, 16), as a GHBP sheddase. TACE has other transmembrane protein substrates, including TNF-, transforming growth factor- (TGF-), and other EGF receptor ligands, TNF receptor-p75, L-selectin, interleukin-6 receptor, and amyloid precursor protein (35, 42, 43). Features in ADAM substrates that allow their proteolysis are poorly understood. In substrates with known cleavage sites, no consensus emerges and cleavage determinants are not simple. The most consistent cleavage site feature among ADAM substrates is that they usually reside in a stalk region between the membrane and an initial globular extracellular subdomain (35). Our findings for the rbGHR cleavage site fit well in this regard with the GHR being an ADAM substrate (34, 44). Indeed, the crystal structure suggests that a short stem of roughly ten residues lies between the transmembrane domain and the beginning of extracellular subdomain 2, which contains the receptor dimerization domain (33).

We exploited the inability of rbGHR-237-239 to undergo cleavage to further examine the relationship between inducible receptor proteolysis and cellular sensitivity to GH. We previously demonstrated such a link in that in several model systems pretreatment with stimuli that induce GHR cleavage lessened the JAK2 activation in response to acute exposure to GH and this desensitization was blocked by inclusion of the metalloprotease inhibitor IC3 during the preincubation (11). In the current study, we found that PMA pretreatment led to desensitization to GH in cells that expressed either WT or rbGHR-237-239-AAA (both cleavable receptors), but not in those that expressed rbGHR-237-239, the non-cleavable receptor. This complements our previous observations gleaned with the metalloprotease inhibitor and strongly implicates metalloprotease-mediated proteolysis as one mechanism of desensitization of GH signaling. Further, the characterization of rbGHR-237-239 as a receptor competent for mediating GH signaling will allow us to pursue studies to better understand how metalloprotease-mediated GHR proteolysis accomplishes desensitization. Using this non-cleavable receptor mutant, we can ask whether it is receptor loss or remnant accumulation that affects signaling by coexpressing with a fixed complement of the non-cleavable receptor varying amounts of a recombinant remnant (engineered to encode residues Phe²³⁹ to 620) and monitoring responsiveness to GH. Such studies should allow us to better address the mechanism(s) by which metalloprotease activity may regulate GH action.

	ACKNOWLEDGEMENTS

We thank Drs. J. Kudlow, J. Messina, K. Zinn, S.-O. Kim, K. Loesch, J. Cowan, and N. Yang for helpful conversations and the generous provision of reagents by those named in the text.

	FOOTNOTES

^* This work was supported by Veterans Affairs Merit Review awards (to S. J. F. and G. B.), a grant from the National Science Foundation (to G. B.), and National Institutes of Health Grants DK46395 and DK58259 (to S. J. F.). Parts of this work were presented at the 84th Annual Endocrine Society Meetings in San Francisco, CA 2002.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: University of Alabama at Birmingham, 1530 3rd Ave. South, BDB 861, Birmingham, AL 35294-0012. Tel.: 205-934-9877; Fax: 205-934-4389; E-mail: frank@endo.dom.uab.edu.

Published, JBC Papers in Press, October 25, 2002, DOI 10.1074/jbc.M208738200

	ABBREVIATIONS

The abbreviations used are: GH, growth hormone; GHR, GH receptor; GHBP, GH-binding protein; Endo H, endoglycosidase H; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; HEK, human embryonic kidney; GFP, green fluorescent protein; WT, wild type; ADAM, a disintegrin and metalloprotease; TACE, tumor necrosis factor--converting enzyme; JAK, Janus-activating kinase; Me₂SO, dimethyl sulfoxide.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				T. M. Piazza, J.-C. Lu, K. C. Carver, and L. A. Schuler Src Family Kinases Accelerate Prolactin Receptor Internalization, Modulating Trafficking and Signaling in Breast Cancer Cells Mol. Endocrinol., February 1, 2009; 23(2): 202 - 212. [Abstract] [Full Text] [PDF]

				X. Wang, J. Jiang, J. Warram, G. Baumann, Y. Gan, R. K. Menon, L. A. Denson, K. R. Zinn, and S. J. Frank Endotoxin-Induced Proteolytic Reduction in Hepatic Growth Hormone (GH) Receptor: A Novel Mechanism for GH Insensitivity Mol. Endocrinol., June 1, 2008; 22(6): 1427 - 1437. [Abstract] [Full Text] [PDF]

				K. Loesch, L. Deng, X. Wang, K. He, J. Jiang, and S. J. Frank Endoplasmic Reticulum-Associated Degradation of Growth Hormone Receptor in Janus Kinase 2-Deficient Cells Endocrinology, December 1, 2007; 148(12): 5955 - 5965. [Abstract] [Full Text] [PDF]

				M. J. van den Eijnden and G. J. Strous Autocrine Growth Hormone: Effects on Growth Hormone Receptor Trafficking and Signaling Mol. Endocrinol., November 1, 2007; 21(11): 2832 - 2846. [Abstract] [Full Text] [PDF]

				S. L. Asa, R. DiGiovanni, J. Jiang, M. L. Ward, K. Loesch, S. Yamada, T. Sano, K. Yoshimoto, S. J. Frank, and S. Ezzat A Growth Hormone Receptor Mutation Impairs Growth Hormone Autofeedback Signaling in Pituitary Tumors Cancer Res., August 1, 2007; 67(15): 7505 - 7511. [Abstract] [Full Text] [PDF]

				N. Yang, X. Wang, J. Jiang, and S. J. Frank Role of the Growth Hormone (GH) Receptor Transmembrane Domain in Receptor Predimerization and GH-Induced Activation Mol. Endocrinol., July 1, 2007; 21(7): 1642 - 1655. [Abstract] [Full Text] [PDF]

				L. Deng, K. He, X. Wang, N. Yang, C. Thangavel, J. Jiang, S. Y. Fuchs, and S. J. Frank Determinants of Growth Hormone Receptor Down-Regulation Mol. Endocrinol., July 1, 2007; 21(7): 1537 - 1551. [Abstract] [Full Text] [PDF]

				E. Bulanova, V. Budagian, E. Duitman, Z. Orinska, H. Krause, R. Ruckert, N. Reiling, and S. Bulfone-Paus Soluble Interleukin (IL)-15R{alpha} Is Generated by Alternative Splicing or Proteolytic Cleavage and Forms Functional Complexes with IL-15 J. Biol. Chem., May 4, 2007; 282(18): 13167 - 13179. [Abstract] [Full Text] [PDF]

				S. Sather, K. D. Kenyon, J. B. Lefkowitz, X. Liang, B. C. Varnum, P. M. Henson, and D. K. Graham A soluble form of the Mer receptor tyrosine kinase inhibits macrophage clearance of apoptotic cells and platelet aggregation Blood, February 1, 2007; 109(3): 1026 - 1033. [Abstract] [Full Text] [PDF]

				S. J. Frank, X. Wang, K. He, N. Yang, P. Fang, R. G. Rosenfeld, V. Hwa, T. R. Chaudhuri, L. Deng, and K. R. Zinn In Vivo Imaging of Hepatic Growth Hormone Signaling Mol. Endocrinol., November 1, 2006; 20(11): 2819 - 2830. [Abstract] [Full Text] [PDF]

				K. Loesch, L. Deng, J. W. Cowan, X. Wang, K. He, J. Jiang, R. A. Black, and S. J. Frank Janus Kinase 2 Influences Growth Hormone Receptor Metalloproteolysis Endocrinology, June 1, 2006; 147(6): 2839 - 2849. [Abstract] [Full Text] [PDF]

				J. L. Bartoe, W. L. McKenna, T. K. Quan, B. K. Stafford, J. A. Moore, J. Xia, K. Takamiya, R. L. Huganir, and L. Hinck Protein interacting with C-kinase 1/protein kinase Calpha-mediated endocytosis converts netrin-1-mediated repulsion to attraction. J. Neurosci., March 22, 2006; 26(12): 3192 - 3205. [Abstract] [Full Text] [PDF]

				A. Flores-Morales, C. J. Greenhalgh, G. Norstedt, and E. Rico-Bautista Negative Regulation of Growth Hormone Receptor Signaling Mol. Endocrinol., February 1, 2006; 20(2): 241 - 253. [Abstract] [Full Text] [PDF]

				V. Budagian, E. Bulanova, Z. Orinska, E. Duitman, K. Brandt, A. Ludwig, D. Hartmann, G. Lemke, P. Saftig, and S. Bulfone-Paus Soluble Axl Is Generated by ADAM10-Dependent Cleavage and Associates with Gas6 in Mouse Serum Mol. Cell. Biol., November 1, 2005; 25(21): 9324 - 9339. [Abstract] [Full Text] [PDF]

				K. He, K. Loesch, J. W. Cowan, X. Li, L. Deng, X. Wang, J. Jiang, and S. J. Frank Janus Kinase 2 Enhances the Stability of the Mature Growth Hormone Receptor Endocrinology, November 1, 2005; 146(11): 4755 - 4765. [Abstract] [Full Text] [PDF]

				D. W. Lambert, M. Yarski, F. J. Warner, P. Thornhill, E. T. Parkin, A. I. Smith, N. M. Hooper, and A. J. Turner Tumor Necrosis Factor-{alpha} Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) J. Biol. Chem., August 26, 2005; 280(34): 30113 - 30119. [Abstract] [Full Text] [PDF]

				J. W. Cowan, X. Wang, R. Guan, K. He, J. Jiang, G. Baumann, R. A. Black, M. S. Wolfe, and S. J. Frank Growth Hormone Receptor Is a Target for Presenilin-dependent {gamma}-Secretase Cleavage J. Biol. Chem., May 13, 2005; 280(19): 19331 - 19342. [Abstract] [Full Text] [PDF]

				J. Jiang, X. Wang, K. He, X. Li, C. Chen, P. P. Sayeski, M. J. Waters, and S. J. Frank A Conformationally Sensitive GHR [Growth Hormone (GH) Receptor] Antibody: Impact on GH Signaling and GHR Proteolysis Mol. Endocrinol., December 1, 2004; 18(12): 2981 - 2996. [Abstract] [Full Text] [PDF]

				E. E. Gardiner, J. F. Arthur, M. L. Kahn, M. C. Berndt, and R. K. Andrews Regulation of platelet membrane levels of glycoprotein VI by a platelet-derived metalloproteinase Blood, December 1, 2004; 104(12): 3611 - 3617. [Abstract] [Full Text] [PDF]

				K.-C. Leung, G. Johannsson, G. M. Leong, and K. K. Y. Ho Estrogen Regulation of Growth Hormone Action Endocr. Rev., October 1, 2004; 25(5): 693 - 721. [Abstract] [Full Text] [PDF]

				V. Budagian, E. Bulanova, Z. Orinska, A. Ludwig, S. Rose-John, P. Saftig, E. C. Borden, and S. Bulfone-Paus Natural Soluble Interleukin-15R{alpha} Is Generated by Cleavage That Involves the Tumor Necrosis Factor-{alpha}-converting Enzyme (TACE/ADAM17) J. Biol. Chem., September 24, 2004; 279(39): 40368 - 40375. [Abstract] [Full Text] [PDF]

				X. Li and H. Fan Loss of Ectodomain Shedding Due to Mutations in the Metalloprotease and Cysteine-rich/Disintegrin Domains of the Tumor Necrosis Factor-{alpha} Converting Enzyme (TACE) J. Biol. Chem., June 25, 2004; 279(26): 27365 - 27375. [Abstract] [Full Text] [PDF]

				X. Wang, K. He, M. Gerhart, J. Jiang, R. J. Paxton, R. K. Menon, R. A. Black, G. Baumann, and S. J. Frank Reduced Proteolysis of Rabbit Growth Hormone (GH) Receptor Substituted with Mouse GH Receptor Cleavage Site Mol. Endocrinol., October 1, 2003; 17(10): 1931 - 1943. [Abstract] [Full Text] [PDF]

				T. E. Golde and C. B. Eckman Physiologic and Pathologic Events Mediated by Intramembranous and Juxtamembranous Proteolysis Sci. Signal., March 4, 2003; 2003(172): re4 - re4. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 277/52/50510    most recent M208738200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, X. Articles by Frank, S. J. Search for Related Content PubMed PubMed Citation Articles by Wang, X. Articles by Frank, S. J. Social Bookmarking                      What's this?