Endocytosis of cell surface receptors is a ubiquitous process that serves multiple functions(1, 2, 3) . Constitutively recycling receptors, such as those for transferrin and low density lipoprotein, are shuttles that deliver their ligands to the cell interior and then return to the plasma membrane. Signaling receptors, on the other hand, are generally not endocytosed efficiently until agonist is bound and transmembrane signaling has occurred. At this point, endocytosis may allow receptor resensitization (e.g. by targeting the receptor to an acidic endosomal compartment that facilitates ligand dissociation, followed by receptor recycling) or may serve as the initial step in the ultimate lysosomal degradation of the receptor (down-regulation)(4) .

For many receptors, efficient endocytosis requires discrete cytoplasmic receptor determinants that serve as signals for interaction with the endocytic machinery of the cell. Positive internalization signals have been identified in constitutively recycling receptors such as transferrin receptors, low density lipoprotein receptors, cation-independent mannose 6-phosphate receptor, and asialoglycoprotein receptors(5, 6) . They are composed of a short linear stretch of cytoplasmic amino acid residues which lack an obvious consensus sequence, but share certain common features. These motifs generally consist of 6 residues that are 20-50 amino acid residues from the plasma membrane and frequently contain an essential tyrosine residue (7) . Such sequences are thought to be directly involved in the association of the receptor with the HA-2 adapter proteins of the membrane clathrin lattice, a process that leads to efficient endocytosis via clathrin-coated pits(8) .

Relatively little is known concerning the molecular basis of endocytosis of heptahelical G-protein-coupled receptors (GPCRs). ()Clathrin-coated pits/vesicles have been implicated as mediating internalization of some(9) , but not other(10, 11) , GPCRs. The sequence NPXXY, similar to the NPXY internalization motif in the low density lipoprotein receptor(5) , is required for efficient agonist-mediated sequestration of the -adrenergic receptor away from the cell surface(12) . This sequence is highly conserved in the seventh membrane-spanning segment of many GPCRs and might serve as a common endocytic signal. Serine/threonine-rich sequences in the third cytoplasmic loop or cytoplasmic tail contribute to efficient internalization of the GPCRs for thyrotropin-releasing hormone(13) , gastrin-releasing peptide(14) , muscarinic agonists(15) , and yeast -factor(16) .

The receptor for parathyroid hormone (PTH) and PTH-related protein (PTHrP) (17) is a member of a newly recognized, structurally distinct heptahelical GPCR subfamily that also includes receptors for secretin, glucagon and related peptides, calcitonin, and several others(18) . Members of this subfamily lack the conserved NPXXY sequence; nonetheless, they undergo efficient ligand-mediated endocytosis. The sequence elements in these receptors required for this process have not been identified. Previous studies of the PTH/PTHrP receptor suggest that endocytosis leading to down-regulation of the receptor occurs in bone and kidney cells in vitro(19, 20) and may serve to regulate target cell responsiveness in vivo(21) .

In the present study, we have used a mutagenesis strategy to identify receptor sequences in the cytoplasmic tail of the PTH/PTHrP receptor that influence receptor-mediated endocytosis. The results indicate the presence of two novel determinants. One is within residues 475-494 and is required for efficient receptor internalization; the second is within residues 459-461, which are highly conserved in the PTH/secretin receptor subfamily, and serves to suppress internalization. These studies illustrate that at least one member of the PTH/secretin receptor subfamily of GPCRs is endocytosed by a signal-dependent mechanism and that such signals can suppress or enhance internalization.

EXPERIMENTAL PROCEDURES

Materials

Mutagenesis by Overlap Extension Polymerase Chain Reaction (PCR)

Internal deletion and tandem alanine mutation constructs were generated by overlap extension PCR, as described by Pease and co-workers(22) . For each receptor construct, four oligonucleotide primers were utilized, two internal primers bearing desired mutation sequences encoding tandem alanines or single alanine and two external primers bearing the 5` end sequences of the final PCR fragment. Primers were designed such that their melting temperatures are between 55 °C and 65 °C. Two overlapping fragments gel-purified from two PCR reactions with paired external and internal primers were mixed for a second PCR reaction. At a low but significant frequency, two overlapping strands were annealed and extended by the DNA polymerase to generate a full-length template which was then amplified by two external primers. The external primer sites are chosen such that the PCR fragment includes a NsiI and SphI sites to facilitate subcloning into the plasmid of interest. The PCR fragments were gel-purified, digested with SphI and NsiI, and ligated with the predigested OKO2 vector. All receptor constructs derived from pcDNA1/AMP (Invitrogen Corp.) were transformed into, maintained, and amplified in E. coli strain, Top10F`. Plasmid DNAs were purified using QIAGEN columns according to the manufacturer's recommended procedure (QIAGEN Corp.). The PCR portions of the final constructs were sequenced to confirm the identity of the PCR product.

Cell Culture and Transfection

PTH/PTHrP Receptor Binding and Internalization

cAMP Levels

RESULTS

Fig. 1presents a diagrammatic representation of the mutant OK PTH/PTHrP receptors encoded by the constructs used in this study. These include a series of C-terminal truncation mutants, designated by ``T'' followed by the residue number of the last amino acid. Also shown are 3 mutants (``D'') in which the residues shown were deleted and replaced by a set of 4 alanine residues.

Figure 1: A schematic diagram of wild type and mutant PTH/PTHrP receptors evaluated in the present study. Progressive truncation at the cytoplasmic tail resulted in mutant PTH/PTHrP receptors (T507, T494, T474, T466, and T458) containing 49, 36, 16, 8, and 0 amino acid residue tails, respectively. In mutant receptors D459-462, D463-466, and D475-494, cytoplasmic tail residues (1-4, 5-8, and 17-36, respectively) were replaced by 4 alanines.

Functional expression of wild-type OK PTH/PTHrP receptors was assessed in COS-7 cells transiently transfected with expression vector OKO2 (Fig. 2). These cells have no detectable endogenous PTH/PTHrP receptors and are thus a suitable model system for these studies. Competitive binding studies (Fig. 2A) demonstrated the presence of PTH/PTHrP receptors that display an apparent affinity (IC) of 4.5 nM, similar to the reported value in an opossum kidney cell line(26) . These receptors were found to be functionally coupled to adenylyl cyclase (Fig. 2B), conferring PTH-dependent increases in cAMP levels, with an EC for bPTH(1-34) of 0.21 nM.

Figure 2: I-labeled PTHrP(1-34)amide binding (A) and bPTH(1-34)-stimulated cAMP production (B) in COS-7 cells transiently expressing the wild-type OK PTH/PTHrP receptor. Binding and cAMP data represent the mean ± S.E. of 8 experiments. Total specific binding was 27 ± 3% of added tracer, and the IC for bPTH(1-34) was 4.5 ± 0.7 nM. Basal and maximal bPTH(1-34)-stimulated cAMP levels were 8 ± 2 and 75 ± 14 (pmol/30-mm well), and the EC for bPTH(1-34) was 0.21 ± 0.03 nM.

Fig. 3compares the kinetics of ligand binding and of receptor internalization in COS-7 cells expressing either wild-type PTH/PTHrP receptors or receptors lacking all but 16 residues in the cytoplasmic tail (T474). These receptors displayed similar ligand binding kinetics (Fig. 3A) and binding affinity (data not shown), but differed markedly in their internalization properties (Fig. 3B). Internalization of the wild-type receptor was rapid (half-maximal within 5 min) and reached a maximal level of 50-60% of cell-associated ligand within 20-30 min. Truncation mutant T474 displayed a similar internalization time course, but only about 25% of cell-associated ligand was internalized after 60 min. To define in more detail the determinants in the cytoplasmic tail required for efficient receptor endocytosis, we evaluated a series of PTH/PTHrP receptor mutants progressively truncated at the C terminus of the cytoplasmic tail (Fig. 4). Functional expression of these mutant receptors, assessed by radioligand binding (Fig. 4A), was variably reduced compared to wild-type for all mutants except T474. No significant differences in binding affinity were seen (data not shown). Results of internalization assays (Fig. 4B) revealed that T507 and T494 were internalized indistinguishably from wild-type, whereas both T474 (as shown above) and T466 were internalized with a 50-60% reduced efficiency.

Figure 3: Time course of PTH/PTHrP receptor internalization in COS-7 cells. The wild-type receptor and mutant T474 were transiently expressed in COS-7 cells, and I-PTHrP(1-34)amide binding (A) and internalization (B) were assessed, as described under ``Experimental Procedures.'' Similar results were obtained in two experiments, each with triplicate data points.

Figure 4: Effects of C-terminal truncation and internal deletion on PTH/PTHrP receptor binding (A) and endocytosis (B). Mutant receptors are as described in Fig. 1. Values are normalized to those of the wild-type receptor which was assigned a value of 100%. Data represent the mean ± S.E. of three to five experiments with triplicate in each. Values with asterisks are significantly different from wild-type (p < 0.005, Student's t test).

These results with truncation mutants suggest the presence within amino acid residues 475-494 of a determinant of efficient PTH/PTHrP receptor endocytosis. However, it is also possible that reduced internalization was due to local effects such as the change in the relative position of the C terminus in T474 and T466. Moreover, as redundant endocytic codes/regions have been identified in a number of receptors(13, 27) , it is also possible that reduced internalization required the deletion of more C-terminal sequence as well as residues 475-494. To investigate these possibilities, an internal deletion mutant (D475-494) in which a sequence region between amino acid residues 475 and 494 was replaced by 4 alanine residues was generated and expressed in COS-7 cells. As shown in Fig. 4B, internalization of this mutant receptor was reduced by 50%, similar to the defect seen with truncation mutant T474. These results indicate that residues 475-494 contain a signal that is required for efficient PTH/PTHrP receptor internalization, and that this signal is functional in the context of the full-length PTH/PTHrP receptor.

Because the truncation mutant T458 was not expressed on the cell surface, it was unclear whether juxtamembrane residues 459-466 contributed to receptor internalization. To investigate this, we generated two tandem alanine mutations targeting this region. The two mutated receptors (D459-462 and D463-466) were expressed at levels somewhat lower than wild-type, based on their ligand binding properties (Fig. 5A). Strikingly, D459-462 displayed a 40% increase in internalization relative to wild-type. D463-466 did not differ from wild-type. These results suggest that the juxtamembrane region of the cytoplasmic tail contains a second endocytic signal that serves to constrain receptor internalization.

Figure 5: Mutations in the juxtamembrane region of the cytoplasmic tail of the PTH/PTHrP receptor: effects on receptor binding (A) and endocytosis (B). Mutant receptors are as described in Fig. 1. Data represent the mean ± S.E. of three experiments with triplicate samples in each. Values with asterisks are significantly different from wild-type (p < 0.005, Student's t test)

Exposure of cells to hypertonic medium has been shown to remove membrane-associated clathrin lattices, resulting in a loss of clathrin-coated pits and vesicles(28) . When COS-7 cells transiently expressing the wild-type OK PTH/PTHrP receptor were exposed to hypertonic solution (0.45 M sucrose) 1 h before the addition of I-PTHrP(1-34)amide, there was an 82% reduction in receptor internalization with no effect on receptor binding per se (Fig. 6). This effect was fully reversible upon restoration of conditions that allow clathrin reassembly (removal of the sucrose). These results are consistent with the notion that ligand-induced PTH/PTHrP receptor internalization occurs via clathrin-coated pits. The internalization of the mutant T474, which is deficient under standard incubation conditions, is further reduced by hypertonic sucrose, indicating that clathrin pit-mediated internalization of the PTH/PTHrP receptor is not solely dependent upon the positive endocytic signal in the tail.

Figure 6: Effect of hypertonic sucrose treatment on PTH/PTHrP receptor binding (A) and endocytosis (B). COS-7 cells transiently expressing the wild-type or the T474 mutant PTH/PTHrP receptor were preincubated ± hypertonic media (0.45 M sucrose) for 1 h, followed by a 1-h incubation with I-PTHrP(1-34) ± 0.45 M sucrose, as indicated. Receptor binding and internalization were then assessed as described under ``Experimental Procedures.'' Values are normalized to those of the wild-type receptor which was assigned a value of 100%. Data represent the mean of two to three independent experiments with triplicate samples in each.

DISCUSSION

Previous studies have suggested that homologous down-regulation of the PTH/PTHrP receptor may limit target cell responsiveness under conditions of ligand excess in vivo(21) , and that this process is associated with receptor internalization(19, 20, 29) . Molecular cloning of the PTH/PTHrP receptor (17) has now allowed us to address the molecular basis of receptor-mediated endocytosis. The results of the present study indicate that the 90 C-terminal amino acid residues of the cytoplasmic tail can be removed without affecting PTH/PTHrP receptor-mediated endocytosis. However, the proximal region of the tail contains two discrete sequence elements that influence internalization, one a positive endocytic signal between residues 17 and 36 of the tail, and the other a negative signal comprising 3 residues at the interface between membrane and cytoplasm.

Two approaches were used to identify the positive endocytic signal. Expression of C-terminal truncation mutants revealed that wild-type levels of receptor-mediated endocytosis were obtained with PTH/PTHrP receptors possessing only a 36amino acid tail. However, further truncation deleting residues LDFKRKARSGSSTYSYGPMV resulted in markedly (50-60%) reduced receptor internalization. Therefore, in the context of a severely truncated PTH/PTHrP receptor, this sequence is sufficient to signal enhanced endocytic efficiency. Moreover, this sequence is required for efficient endocytosis since a nontruncated receptor in which this sequence was specifically deleted displayed comparably reduced endocytosis.

The relationship between the signal contained within this sequence and previously identified positive endocytic signals is unclear. The sequence does not contain a heretofore identified internalization signal, but resembles known signals in certain respects. It is rich in serine/threonine residues as are internalization sequences in the cytoplasmic tails of the thyrotropin-releasing hormone(13) , gastrin-releasing peptide(14) , and yeast -factor (16) receptors. Recently, we have found that the cytoplasmic tail of the PTH/PTHrP receptor is rapidly phosphorylated upon agonist binding, ()a modification that could promote receptor internalization. Also present are 2 tyrosine residues in a stretch of sequence likely to have -turn structure by Chou-Fasman analysis(30) . A tyrosine residue in the context of a -turn is a characteristic feature of internalization signals in several constitutively recycling receptors (7) and could play a similar role in signaling receptors. In this regard, preliminary mutagenesis studies indicate that the positive signal is contained within the sequence TYSYGPMV. ()This stretch contains 2 tyrosines included in the sequence TYSYG which has a high probability of forming a turn(30) . Unlike most of the cytoplasmic tail, this sequence is highly conserved among PTH/PTHrP receptors from opossum to human(18) , suggesting a functional role. Further mutagenesis should serve to clarify the role of serine/threonine phosphorylation and/or tyrosine residues in the endocytic function of this domain.

Mutated PTH/PTHrP receptors lacking the positive internalization signal (e.g. T474) are endocytosed, albeit to a reduced extent. This is not due entirely to bulk flow internalization, as disruption of clathrin lattices by treatment with hypertonic sucrose further suppressed internalization. The present signal may act in concert with one or more additional positive endocytic signals located outside of the receptor's cytoplasmic tail. It will be important to establish whether this signal can function autonomously to promote endocytosis, e.g. when spliced onto an internalization-defective transferrin receptor.

An important issue for signaling receptors is the relationship between signal transduction and receptor internalization. In the case of epidermal growth factor and insulin receptors, mutations that abolish intrinsic tyrosine kinase activity markedly reduce receptor-mediated endocytosis(31, 32) . In the case of the epidermal growth factor receptor, tyrosine kinase activity, but not autophosphorylation, is required, indicating that signal transduction and heterologous protein phosphorylation is a prerequisite for efficient internalization(32, 33) . For GPCRs, endocytic signals may be found in cytoplasmic domains that are either independent of (34) or overlap with (35) regions that are required for agonist-stimulated signal transduction. In the present study, truncation mutants lacking the positive endocytic signal appear to function similarly to wild-type with respect to adenylyl cyclase activation, indicating that internalization can be disrupted in mutants that retain the ability to signal adenylyl cyclase. We do not yet know whether the results of transmembrane signaling (e.g. activation of protein kinase A or protein kinase C) are involved in regulating PTH/PTHrP receptor endocytosis.

Tandem alanine mutagenesis of the proximal region of the cytoplasmic tail revealed a second domain that influences PTH/PTHrP receptor-mediated endocytosis. Mutation D459-461, in which residues EVQ at the membrane-cytoplasmic interface were mutated to AAA, displayed a marked, 40% increase in internalization. This result suggests that the EVQ sequence serves to limit the entry of receptors into endocytosis-competent structures, either by facilitating interaction of the receptor with poorly endocytosed membrane components or by directly inhibiting the function of a positive endocytic signal(s). It is noteworthy that this EVQ sequence is highly conserved across members of the PTH/secretin receptor subfamily (but not in other GPCRs) and may serve as a suppressor of endocytosis in other subfamily members. Although the vast majority of endocytic signals previously described are positive signals, a juxtamembrane cytoplasmic domain that inhibits coated-pit localization and endocytosis has been identified in an isoform of the Fc receptor(36) . In that case, the minimum sequence required for inhibition is not yet clear. Rodriguez et al.(37) found that truncation of the distal portion of the cytoplasmic tail of the luteinizing hormone/human choriogonadotropin receptor resulted in enhanced receptor-mediated hormone internalization, suggesting the removal of a negative endocytic signal. Thus, these results and ours indicate that receptor-mediated endocytosis is a highly complex and regulated process, with multiple receptor domains playing facilitory as well as inhibitory roles.

Both clathrin-coated and noncoated endocytic pathways have been suggested for GPCRs. For example, -adrenergic receptors have been reported to be internalized through noncoated vesicles(11) . However, a recent report showed that -adrenergic receptors co-localized in endosomes with transferrin, which is known to be internalized in clathrin-coated pits/vesicles(38) . It is not clear whether this discrepancy is due to differences in the cell lines used or whether noncoated vesicles are able to fuse with clathrin-coated vesicles. The marked inhibition of PTH/PTHrP receptor internalization produced by hypertonic sucrose, a treatment known to disrupt clathrin lattices (28) , suggests that PTH/PTHrP receptor endocytosis occurs at least in part through a clathrin-coated pit mechanism. This result is consistent with the previous report of Silve et al.(39) who used EM autoradiography to demonstrate the presence of iodinated PTH in clathrin-coated pits on the surface of cells in calvarial bone.

In summary, specific positive and negative signals in the cytoplasmic tail of a GPCR, the PTH/PTHrP receptor, serve to regulate agonist-stimulated endocytosis via clathrin-coated pits/vesicles. These signals are distinct from those previously identified in both constitutively recycling and signaling receptors. Further delineation of the critical features of these signals and identification of their targets should provide new insights into the molecular basis of endocytosis of the PTH/PTHrP receptor and possibly other GPCRs.

FOOTNOTES

*

This work was supported by the Medical Research Service of the Department of Veterans Affairs and by National Institutes of Health Grant DK35323. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Research Career Scientist of the Department of Veterans Affairs. To whom correspondence and reprint requests should be addressed. Tel.: 415-750-2089; Fax: 415-750-6929.

()

The abbreviations used are: GPCR, G-protein-coupled receptor; PTH, parathyroid hormone; bPTH, bovine PTH; PTHrP, parathyroid hormone-related peptide; PCR, polymerase chain reaction; OK, opossum kidney.

()

E. Blind and R. A. Nissenson, submitted for publication.

()

Z. Huang and R. A. Nissenson, unpublished data.

ACKNOWLEDGEMENTS

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				S. L. Ferrari and A. Bisello Cellular Distribution of Constitutively Active Mutant Parathyroid Hormone (PTH)/PTH-Related Protein Receptors and Regulation of Cyclic Adenosine 3',5'-Monophosphate Signaling by {beta}-Arrestin2 Mol. Endocrinol., January 1, 2001; 15(1): 149 - 163. [Abstract] [Full Text]

				K. N. Pandey, R. Kumar, M. Li, and H. Nguyen Functional Domains and Expression of Truncated Atrial Natriuretic Peptide Receptor-A: The Carboxyl-Terminal Regions Direct the Receptor Internalization and Sequestration in COS-7 Cells Mol. Pharmacol., February 1, 2000; 57(2): 259 - 267. [Abstract] [Full Text]

				K. Nakamura, X. Liu, and M. Ascoli Seven Non-contiguous Intracellular Residues of the Lutropin/Choriogonadotropin Receptor Dictate the Rate of Agonist-induced Internalization and Its Sensitivity to Non-visual Arrestins J. Biol. Chem., January 7, 2000; 275(1): 241 - 247. [Abstract] [Full Text] [PDF]

				S. L. Ferrari, V. Behar, M. Chorev, M. Rosenblatt, and A. Bisello Endocytosis of Ligand-Human Parathyroid Hormone Receptor 1 Complexes Is Protein Kinase C-dependent and Involves beta -Arrestin2. REAL-TIME MONITORING BY FLUORESCENCE MICROSCOPY J. Biol. Chem., October 15, 1999; 274(42): 29968 - 29975. [Abstract] [Full Text] [PDF]

				C. Baratti-Elbaz, N. Ghinea, O. Lahuna, H. Loosfelt, C. Pichon, and E. Milgrom Internalization and Recycling Pathways of the Thyrotropin Receptor Mol. Endocrinol., October 1, 1999; 13(10): 1751 - 1765. [Abstract] [Full Text]

				S. Hoare, J. A. Copland, Z. Strakova, K. Ives, Y.-J. Jeng, M. R. Hellmich, and M. S. Soloff The Proximal Portion of the COOH Terminus of the Oxytocin Receptor Is Required for Coupling to Gq, but Not Gi. INDEPENDENT MECHANISMS FOR ELEVATING INTRACELLULAR CALCIUM CONCENTRATIONS FROM INTRACELLULAR STORES J. Biol. Chem., October 1, 1999; 274(40): 28682 - 28689. [Abstract] [Full Text] [PDF]

				K. Nakamura, X. Liu, and M. Ascoli The Rate of Internalization of the Gonadotropin Receptors Is Greatly Affected by the Origin of the Extracellular Domain J. Biol. Chem., September 3, 1999; 274(36): 25426 - 25432. [Abstract] [Full Text] [PDF]

				M. B. Wheeler, R. W. Gelling, S. A. Hinke, B. Tu, R. A. Pederson, F. Lynn, J. Ehses, and C. H. S. McIntosh Characterization of the Carboxyl-terminal Domain of the Rat Glucose-dependent Insulinotropic Polypeptide (GIP) Receptor. A ROLE FOR SERINES 426 AND 427 IN REGULATING THE RATE OF INTERNALIZATION J. Biol. Chem., August 27, 1999; 274(35): 24593 - 24601. [Abstract] [Full Text] [PDF]

				A. M. Cypess, C. G. Unson, C.-R. Wu, and T. P. Sakmar Two Cytoplasmic Loops of the Glucagon Receptor Are Required to Elevate cAMP or Intracellular Calcium J. Biol. Chem., July 2, 1999; 274(27): 19455 - 19464. [Abstract] [Full Text] [PDF]

				Z. Huang, T. Bambino, Y. Chen, J. Lameh, and R. A. Nissenson Role of Signal Transduction in Internalization of the G Protein-Coupled Receptor for Parathyroid Hormone (PTH) and PTH-Related Protein Endocrinology, March 1, 1999; 140(3): 1294 - 1300. [Abstract] [Full Text]

				J. Hilpert, A. Nykjaer, C. Jacobsen, G. Wallukat, R. Nielsen, S. K. Moestrup, H. Haller, F. C. Luft, E. I. Christensen, and T. E. Willnow Megalin Antagonizes Activation of the Parathyroid Hormone Receptor J. Biol. Chem., February 26, 1999; 274(9): 5620 - 5625. [Abstract] [Full Text] [PDF]

				G. N. Prado, D. F. Mierke, M. Pellegrini, L. Taylor, and P. Polgar Motif Mutation of Bradykinin B2 Receptor Second Intracellular Loop and Proximal C Terminus Is Critical for Signal Transduction, Internalization, and Resensitization J. Biol. Chem., December 11, 1998; 273(50): 33548 - 33555. [Abstract] [Full Text] [PDF]

				N. Malecz, T. Bambino, M. Bencsik, and R. A. Nissenson Identification of Phosphorylation Sites in the G Protein-Coupled Receptor for Parathyroid Hormone. Receptor Phosphorylation Is Not Required for Agonist-Induced Internalization Mol. Endocrinol., December 1, 1998; 12(12): 1846 - 1856. [Abstract] [Full Text]

				N. Hukovic, R. Panetta, U. Kumar, M. Rocheville, and Y. C. Patel The Cytoplasmic Tail of the Human Somatostatin Receptor Type 5 Is Crucial for Interaction with Adenylyl Cyclase and in Mediating Desensitization and Internalization J. Biol. Chem., August 14, 1998; 273(33): 21416 - 21422. [Abstract] [Full Text] [PDF]

				I. Ishii, E. Saito, T. Izumi, M. Ui, and T. Shimizu Agonist-induced Sequestration, Recycling, and Resensitization of Platelet-activating Factor Receptor. ROLE OF CYTOPLASMIC TAIL PHOSPHORYLATION IN EACH PROCESS J. Biol. Chem., April 17, 1998; 273(16): 9878 - 9885. [Abstract] [Full Text] [PDF]