Requirement of tyrosine residues 333 and 338 of the growth hormone (GH) receptor for selected GH-stimulated function.

We have examined the involvement of tyrosine residues 333 and 338 of the growth hormone (GH) receptor in the cellular response to GH. Stable Chinese hamster ovary (CHO) cell clones expressing a receptor with tyrosine residues at position 333 and 338 of the receptor substituted for phenylalanine (CHO-GHRY333F, Y338F) were generated by cDNA transfection. Compared with the wild type receptor the Y333F,Y338F mutant possessed normal high affinity ligand binding, hormone internalization, and ligand-induced receptor down-regulation. GH activation of mitogen-associated protein kinase was also similar in CHO clones expressing similar wild type and Y333F,Y338F receptor number. However, two GH-regulated cellular events (lipogenesis, and protein synthesis) were deficient in the tyrosine substituted receptor. In contrast, transcriptional regulation by GH (as evidenced by chloramphenicol acetyltransferase cDNA expression driven by the GH-responsive region of the SPI 2.1 gene) was not affected by Y333F,Y338F substitution. Thus we provide the first experimental evidence that specific tyrosine residues of the GH receptor are required for selected cellular responses to GH.

We have examined the involvement of tyrosine residues 333 and 338 of the growth hormone (GH) receptor in the cellular response to GH. Stable Chinese hamster ovary (CHO) cell clones expressing a receptor with tyrosine residues at position 333 and 338 of the receptor substituted for phenylalanine (CHO-GHR 1-638 Y333F, Y338F) were generated by cDNA transfection. Compared with the wild type receptor the Y333F,Y338F mutant possessed normal high affinity ligand binding, hormone internalization, and ligand-induced receptor down-regulation. GH activation of mitogen-associated protein kinase was also similar in CHO clones expressing similar wild type and Y333F,Y338F receptor number. However, two GH-regulated cellular events (lipogenesis, and protein synthesis) were deficient in the tyrosine substituted receptor. In contrast, transcriptional regulation by GH (as evidenced by chloramphenicol acetyltransferase cDNA expression driven by the GH-responsive region of the SPI 2.1 gene) was not affected by Y333F,Y338F substitution. Thus we provide the first experimental evidence that specific tyrosine residues of the GH receptor are required for selected cellular responses to GH.
The GH 1 receptor is a member of the cytokine receptor superfamily (Bazan, 1990;Cosman et al., 1990;Kitamura et al., 1994). One characteristic of this family of receptors is the lack of a canonical sequence for intrinsic tyrosine kinase activity (Leung et al., 1987;Mathews et al., 1989;Smith et al., 1989;Kitamura et al., 1994). However, ligand binding to a number of these receptors, including the GH receptor, induces tyrosyl phosphorylation of cellular proteins (Möller et al., 1992;Wang et al., 1992Wang et al., , 1993. This tyrosyl phosphorylation of cellular proteins is attributed to the association with Janus kinase 2 (JAK2) for the GH receptor (Argetsinger et al., 1993) and also the receptors for erythropoietin, prolactin, interleukin-3, interleukin-6, granulocyte-colony-stimulating factor, granulocytemacrophage colony-stimulating factor, leukocyte inhibitory factor, oncostatin M, and ciliary neurotrophic factor Stahl et al., 1994;Narazaki et al., 1994;Dusanter-Fourt et al., 1994;DaSilva et al., 1994). Pharmacological inhibition of cellular tyrosine phosphorylation does inhibit certain GH-stimulated cellular events Möller et al., 1994;Sliva et al., 1994). Although the GH receptor is itself tyrosine-phosphorylated (Foster et al., 1988), it has not been demonstrated that such receptor phosphorylation is required for the biological response to GH.
Studies involving truncation of the GH receptor intracellular domain has suggested functional specificity for different regions of the intracellular domain. Thus the membrane proximal portion alone (truncated at amino acid 454) is capable of mediation of GH-stimulated mitogenesis (Möller et al., 1992;Colosi et al., 1993), lipid synthesis , protein synthesis , MAP kinase activation (Möller et al., 1992), c-fos expression (VanderKuur et al., 1994) and hormone internalization (Möldrup et al., 1991). In contrast the carboxyl-terminal portion of the GH receptor is required for full transcriptional activation (Goujon et al., 1994) and phosphorylation of p97 (a putative GH-dependent transcription factor) (VanderKuur et al., 1994). The membrane proximal portion of the intracellular domain contains 4 tyrosine residues but a single set of paired tyrosine residues at position 333 and 338 of the receptor. Phosphorylation of specific tyrosyl residues in receptor molecules serve as docking sites for for one or more proteins containing SH2 domains (Koch et al., 1991;Cantley et al., 1991;Mayer and Baltimore, 1993;Nishimura et al., 1993). Concordant with functional specificity of other receptor molecules being determined by phosphorylation of specific tyrosine residues (Mohammadi et al., 1992;Pawson and Gish, 1992), we have investigated the importance of tyrosine 333 and 338 of the GH receptor for the biologic response to GH. We show here that phosphorylation of tyrosine 333 and 338 are required for selected biological functions of GH.  sham, United Kingdom). Triton X-100, dexamethazone, myelin basic protein, and BSA were from Sigma. The ␤-galactosidase expression vector (pCH110) was obtained from Pharmacia (Uppsala, Sweden). MAP kinase monoclonal antibody was purchased from Zymed Laboratories, Inc. (S. San Francisco, CA). All other reagents were of reagent grade or higher.
Cellular Transfection-Rat GH receptor cDNA was cloned into an expression plasmid containing an SV40 enhancer and a human metallothionein IIa promoter. The cDNAs were transfected into CHO-K1 cells with Lipofectin together with the pIPB-1 plasmid which contains a neomycin resistance gene fused to the thymidine kinase promoter. Stable integrants were selected using 1000 g/ml G418. The complete rat GH receptor cDNA (Mathews et al., 1989) coding for amino acids 1-638 was expressed in CHO4 -638 (Emtner et al., 1990) or CHOA-638 cells (CHO-GHR 1-638 ) (Wang et al., 1993). A stop codon was created at amino acid 295 by in vitro mutagenesis of GH receptor cDNA to create a membrane-bound, but cytoplasmic domain-deficient, receptor. This cDNA was expressed in CHO-294 (CHO-GHR 1-294 ) cells (Möller et al., 1992). The Y333F,Y338F mutant cDNA was constructed using polymerase chain reaction as described. 2 Oligonucleotides carrying the substitution were synthesized and used as primers in order to introduce point mutations. The introduced mutation was confirmed by DNA sequence analysis. This cDNA was expressed in CHO-GHR 1-638 Y333F,Y338F cells (VanderKuur et al., 1995b).
Hormone Internalization, Degradation, and Receptor Turnover-CHO cells were plated in six-well plates (Moldrup et al., 1991). Cell monolayers were washed in HEPES binding buffer (10 mM HEPES, pH 7.4, 124 mM NaCl, 4 mM CaCl 2 , 1.5 mM MgCl 2 ) and incubated on ice for 3 h in 1 ml of binding buffer containing 100,000 cpm 125 I-hGH. The medium was removed, replaced with HEPES binding buffer, the cells rapidly warmed and incubated at 37°C for the indicated time. The cells were again placed on ice, the incubation medium was removed, and the cells were washed four times with ice-cold HEPES binding buffer. Internalized GH was determined by acid wash (0.15 M NaCl, 0.05 M glycine, pH 2.5) and measurement of the cell-associated radioactivity after solubilization with 1 M NaOH. The amount of degraded (trichloroacetic acid-soluble) and intact (trichloroacetic acid-precipitable) was determined by precipitation in 10% trichloroacetic acid and quantitation of radioactivity in the pellet and supernatant.
For estimation of ligand-induced receptor down-regulation, cell monolayers in six-well plates were incubated in the presence of different concentrations of hGH in serum-free medium for 12 h (Roupas and Herington, 1988). The cells were placed on ice and associated hGH removed by acid wash (0.15 M NaCl, 0.05 M glycine, pH 2.5) and further washed three times in a wash buffer containing 10 mM HEPES, pH 7.4, 124 mM NaCl, 4 mM CaCl 2 , and 1.5 mM MgCl 2 . The cells were then incubated for 90 min at room temperature in binding medium (10 mM HEPES, pH 7.4, 1% human serum albumin) containing 125 I-hGH (approximately 100,000 cpm). The cells were washed four times in wash buffer and solubilized with 1 ml of 1 M NaOH. Radioactivity was counted in a ␥ counter. Nonspecific binding was estimated in the presence of 1 g/ml unlabeled hGH and was Ͻ5%.
MAP Kinase Activity-Cells were cultured in 100-mm dishes to near confluence and serum-deprived for 12 h before stimulation with 20 nM hGH. MAP kinase activity was measured in immunoprecipitates of solubilized cells essentially as described (Ahn et al., 1990). Briefly, cells were solubilized in 2 ml of buffer containing 50 mM Tris-HCl, pH 7.4, 1.5 mM EGTA, 10 mM MgCl 2 , 0.1 mM Na 3 VO 4 and 1% Triton X-100. Particulate material was removed by centrifugation at 20,000 ϫ g for 10 min. The supernatant was used for immunoprecipitation using 0.5 g/ml of monoclonal MAP kinase antibody. In vitro phosphorylation was performed using myelin basic protein as substrate. The reaction mixture was spotted onto Whatman 81 paper, washed in 10% trichloroacetic acid, rinsed in ethanol, and counted by liquid scintillation.
Lipid Synthesis-Lipogenesis was estimated as described by Moody et al. (1974) with minor modification. Cells were grown to confluence in six-well plates and incubated in serum-free medium (Ham's F-12) supplemented with 1% (w/v) BSA for 12 h. The assay was initiated by the addition of 1 ml of Krebs-Ringer-HEPES buffer containing 1% (w/v) BSA, 0.55 mM glucose, 0.5 Ci of [ 3 H]glucose (5-15 Ci/mmol) Ϯ hGH (100 nM). The incubation was continued for 2 h and terminated by washing with cold phosphate-buffered saline followed by the addition of 0.5 ml of 0.5 M NaOH, 0.1% (v/v) Triton X-100. Solubilized cells were transferred to scintillation vials containing 3.5 ml of a toluene scintillant, and lipid-incorporated radioactivity was measured in the organic phase by scintillation counting. Results are expressed as the percentage induction above control, where cells were incubated in the absence of hGH.
Protein Synthesis-Cells were grown to confluence in 60-mm cell culture dishes and washed with phosphate-buffered saline and incubated in serum-free media (Ham's F-12) for 24 h. The cells were washed once more in serum-free medium before incubation with 100 nM hGH for 12 h. Protein synthesis was estimated by incorporation of [ 3 H]leucine into trichloroacetic acid-precipitable proteins (Emtner et al., 1990). The cells were pulse-labeled with 3 Ci of [ 3 H]leucine for 2 h, washed four times with cold phosphate-buffered saline, and solubilized in 2 ml of 0.1% SDS. Protein was precipitated by the addition of 200 l of trichloroacetic acid and incubation for 60 min at 4°C. The precipitate was collected on glass fiber filters and the filters washed twice with 4% trichloroacetic acid and 100% ethanol (2 ml). Radioactivity on the filters was quantified by liquid scintillation counting. Results are presented as the -fold stimulation above cells assayed in the absence of hGH.
Transient Transfection of SPI-CAT Reporter Constructs-Cells were grown to 50% confluence in 60-mm dishes. Twenty-four hours prior to transfection, the cells were washed twice with Dulbecco's modified Eagle's medium and serum-free medium consisting of a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 supplemented with 10 g/ml of transferrin, 80 milliunits/ml of insulin, 2.5 mM glutamine, and nonessential amino acids. CHO cells were transiently transfected by the calcium phosphate procedure with 3 g of pCH110 (␤galactosidase expression vector, Pharmacia, Uppsala, Sweden), 1.5 g of the construct containing the bacterial chloramphenicol acetyltransferase (CAT) coding sequence linked to Ϫ149 to Ϫ103 of the serine protease inhibitor (SPI 2.1) gene promotor (Yoon et al., 1990) and 3 g of the Y333F,Y338F GH receptor or wild type receptor plasmids. After the cells were subjected to glycerol shock, fresh GC3 medium with dexamethazone (10 M) was added Ϯ hGH. Cells were scraped from the plate after 48 h, and extracts were prepared by three consecutive freeze-thaw cycles followed by centrifugation at 15,000 ϫ g for 10 min. Aliquots of the supernatant were normalized for ␤-galactosidase activity and then assayed for CAT activity. CAT assays were performed as described with 1 Ci of [ 14 C]chloramphenicol. The samples were subjected to thin layer chromatography and exposed to PhosphorImager screens (Molecular Dynamics, Sunnyvale, CA). CAT activity was determined by quantification of the spots corresponding to [ 14 C]chloramphenicol and its acetylated forms.
Hormone Internalization and Degradation in CHO-GHR 1-638 Y333F,Y338F-GH is subject to high efficiency internalization upon stable transfection of CHO cells with wild type GH receptor cDNA Lobie et al., 1994a). We therefore examined whether Tyr 333 and Tyr 338 of the GH receptor were involved in receptor-mediated hormone internalization. As observed in Fig. 1 the hormone internalization kinetics for both full-length receptor and the Y333F,Y338F mutated receptor were identical. Furthermore, no differences in hormone degradation were observed between stable cell clones expressing the full-length receptor or the Y333F,Y338F mutation.
Ligand-induced Receptor Down-regulation in CHO-GHR 1-638 Y333F,Y338F-The GH receptor is subject to ligandinduced down-regulation and subsequent lysosomal destruction (Roupas and Herington, 1988). We therefore examined whether Y333F,Y338F substitution of the GH receptor affected ligand-dependent receptor down-regulation. As observed in Fig. 2, CHO cells transfected with the full-length receptor exhibited the expected dose-dependent receptor down-regulation. CHO-GHR 1-638 Y333F,Y338F clone 23 exhibited identical receptor down-regulation, indicative that Tyr 333 and Tyr 338 are not involved in ligand-induced receptor down-regulation.
Activation of MAP Kinase in CHO-GHR 1-638 Y333F,-Y338F-We have demonstrated previously that GH increases MAP kinase activity in CHO cells transfected with cDNA for the full-length GH receptor (Möller et al., 1992). GH is also able to activate MAP kinase via a receptor lacking the carboxylterminal half of the receptor intracellular domain (Möller et al., 1992;VanderKuur et al., 1994). We examined the possibility that Y333F,Y338F substitution was involved in the activation of MAP kinase. GH stimulation of MAP kinase in CHO-GHR 1-638Y333F,Y338F clone 3 was significantly less than that observed in CHO-GHR 1-638 . However, when CHO-GHR 1-638Y333F,Y338F clone 23 with a higher receptor number was used the activation of MAP kinase activity was comparable with that observed with the wild type receptor (Fig. 3). Thus Tyr 333 and Tyr 338 of the GH receptor are not involved in the activation of MAP kinase by GH.
Protein Synthesis in CHO-GHR 1-638 Y333F,Y338F-Growth hormone has been demonstrated previously to increase cellular protein synthesis. This effect is reconstituted in GH receptor cDNA-transfected cells and GH-stimulated protein synthesis does not require the carboxyl-terminal half of the receptor intracellular domain (Emtner et al., 1990;Billestrup et al., 1994). We, therefore, examined the effect of phenylalanine substitution of tyrosine at positions 333 and 338 of the receptor on the GH stimulation of protein synthesis. This was compared with cells stably transfected with cDNAs for the wild type receptor (CHO-GHR 1-638 ) and a membrane-bound, but cytoplasmic domain-deficient, receptor (CHO-GHR 1-294 ) (for transfection control). GH treatment of CHO-GHR 1-638 cells resulted in a 1.6-fold increase in the total cellular protein synthesis. In contrast neither of the CHO-GHR 1-638 Y333F,Y338F clones nor CHO-GHR 1-294 cells displayed any significant enhancement of protein synthesis upon GH stimulation (Fig. 4).
Lipogenesis in CHO-GHR 1-638 Y333F,Y338F-GH has also been demonstrated previously to stimulate lipogenesis in GH receptor cDNA-transfected cells . This effect also does not require the carboxyl-terminal half of the receptor intracellular domain. We therefore examined GH-stimulated lipogenesis in the different CHO stable transfectants. GH treatment of CHO-GHR 1-638 cells resulted in a 2-fold increase in cellular lipogenesis within 2 h after addition of hormone. Neither of the CHO-GHR 1-638 Y333F,Y338F clones nor CHO-GHR 1-294 cells displayed any significant enhancement of lipid synthesis upon GH stimulation (Fig. 5).
SPI-CAT Expression in CHO-GHR 1-638 Y333F,Y338F-GH has been demonstrated previously to drive CAT cDNA expression in reporter constructs containing multiple repeats of a 45-base pair GH-responsive region of the SPI 2.1 gene promotor (Yoon et al., 1990;Enberg et al., 1994;Goujon et al., 1994;Silva et al., 1994). These transcriptional regulatory effects of GH require the entire intracellular domain (Enberg et al., 1994;Goujon et al., 1994). This is in contrast to GH-stimulated lipogenesis, protein synthesis, mitogenesis, and MAP kinase activation which require only the membrane proximal portion of the intracellular domain. We therefore examined the ability of GH to stimulate transcription via the Y333F,Y338F receptor. As is evident from Fig. 6 both the wild type receptor and the Y333F,Y338F receptor drive CAT expression to a similar extent. Thus Tyr 333 and Tyr 338 of the GH receptor are not involved in GH regulation of transcription. (GHR 1-638 ) and for the Y333F,Y338F receptor (GHR 1-638 Y333F,Y338F). Estimation of receptor down-regulation was performed as described under "Experimental Procedures" using 125 I-hGH (approximately 100,000 cpm). Results are presented as the mean Ϯ S.E. of quadruplicate determinations. Data shown are derived from CHOA cells (GHR 1-638 ) and GHR 1-638 Y333F,Y338F clone 23. Similar results were obtained with the use of CHO4 cells and clone 3 of GHR 1-638 Y333F,Y338F.

FIG. 3. GH activation of MAP kinase activity in CHO cells and in CHO cells stably transfected with cDNA for the wild type
GH receptor (GHR 1-638 ) and for the Y333F,Y338F receptor (GHR 1-638 Y333F,Y338F (clones 3 and 23)). MAP kinase activity in immunoprecipitates of MAP kinase was analyzed 15 min after stimulation with GH using myelin basic protein as substrate. Results are presented as mean Ϯ S.D. from triplicate determinations. Results for GHR 1-638 presented are derived from CHOA cells. Similar results were obtained with CHO4 cells and also have been reported previously (Möller et al., 1992). DISCUSSION We demonstrate here that specific tyrosine residues within the GH receptor are required for GH to elicit selective cellular function. We refer specifically to Tyr 333 and Tyr 338 of the GH receptor and their involvement in GH-mediated protein synthesis and lipogenesis. The selective retention of certain GHstimulated functions within the cell (also see VanderKuur et al., 1995b) further substantiates the specific involvement of these residues in GH signal transduction.
Several studies have implicated the existence of distinct receptor domains regulating GH-stimulated events (Moldrup et al., 1991;Möller et al., 1992Möller et al., , 1994Colosi et al., 1993;Enberg et al., 1994;Goujon et al., 1994;VanderKuur et al., 1994). Truncation of the intracellular domain of the receptor has allowed definition of two distinct macrodomains within the receptor with differential involvement in transcription and metabolic events. A complete transcriptional response to GH requires the presence of the carboxyl-terminal half of the intracellular domain. In contrast certain presumed "metabolic" events such as mitogenesis lipogenesis and protein synthesis are mediated by the membrane proximal portion of the intracellular domain. That specific tyrosine residues are necessary for selected functions further reinforces the concept of distinct receptor domains. However, since no absolute delineation exists (e.g. both MAP kinase activation and protein synthesis require only the membrane-proximal portion of the intracellular domain, yet only protein synthesis is deficient in the Y333F,Y338F receptor), then regional functional specificity of the receptor is likely to be dictated by the association of specific signaling molecules to discrete phosphorylated tyrosine residues. Such is the case for the better characterized epidermal growth factor and plate-let-derived growth factor receptors, where specific phosphorylated tyrosine residues serve as docking sites for proteins containing SH2 domains (Nishimura et al., 1993). That transcriptional up-regulation is not affected by Y333F,Y338F substitution in the receptor indicates that some more distal CHO cells were transiently transfected by the calcium phosphate procedure and also with a construct containing bacterial CAT coding sequence linked to Ϫ149/Ϫ103 of the serine protease inhibitor (SPI 2.1) gene promotor. The cells were processed, and CAT assays were performed as described under "Experimental Procedures." Results are mean Ϯ S.D. from triplicate determinations. tyrosine residue(s) may be involved in this event. GH has recently been demonstrated to activate transcription by phosphorylation of a factor binding to a GAS-like DNA element (Meyer et al., 1994;Finbloom et al., 1994;Sliva et al., 1994). It has been reported that this factor is p91 (STAT1)-and STAT3-like for the SIE element (Meyer et al., 1994;Campbell et al., 1995) and STAT5 like for the SPI 2.1 GAS-like element (Wood et al., 1995). The ␥-interferon receptor requires a distal tyrosine residue (Tyr 440 ) for activation of STAT 1 and presumably functions through direct receptor-STAT1 interaction (Greenlund et al., 1994). The GH receptor could also hypothetically participate in transcriptional activation by providing a distal tyrosine residue for docking and subsequent phosphorylation of the STAT protein. Also receptor dimerization (Cunningham et al., 1991;DeVos et al., 1992), presumably after ligand binding, could present a conformation that allows docking of signaling molecules regardless of the phosphorylation state. At present we do not know the nature of the molecular interaction(s) that is potentially disrupted by the Y333F,Y338F substitution. These tyrosines are not part of a known sequence reported to serve as docking sites for signaling molecules (Cantley et al., 1991).
The GH stimulation of protein synthesis does not require RNA synthesis (Martin and Young, 1965). Instead GH treatment of animals is associated with an apparent increase in ribosomal efficiency (Kostyo and Rilemma, 1971). Increase in cellular protein synthesis is thought to be mediated by phosphorylation of the ribosomal protein S6. The phosphorylation of ribosomal protein S6 is mediated by S6 kinase, which is itself phosphorylated by MAP kinase (Maller, 1991). GH has been reported to activate both MAP kinase (Winston and Bertics, 1992;Campbell et al., 1992;Möller et al., 1992) and S6 kinase (Anderson, 1993). That protein synthesis was deficient in the Y333F,Y338F receptor indicates that simple MAP kinase activation is also insufficient for the GH induction of protein synthesis. This is interesting as S6 kinase is reported to only partially phosphorylate ribosomal protein S6, with additional phosphorylation mediated by cAMP-dependent protein kinase (Palen and Traugh, 1987). There are reports that GH may interact with the cAMP pathway (Catalioto et al., 1992;Singh and Thomas, 1993). Further activation of S6 kinase can be mediated by IRS-1 (Myers et al., 1994), and IRS-1 has been demonstrated to be phosphorylated in response to GH (Souza et al., 1994;Ridderstråle et al., 1995;Argetsinger et al., 1995). It has been demonstrated recently that IL-3 stimulation of protein synthesis is mediated by the regulation of double-stranded RNA-dependent protein kinase (Ito et al., 1994). This molecule inhibits protein synthesis by phosphorylating the ␣ subunit of eucaryotic transcription factor 2. Such may also be the case for GH. It is possible that MAP kinase is only involved in a negative regulatory feed back loop. Indeed MAP kinase has been demonstrated to decrease epidermal growth factor receptor phosphorylation by activation of a tyrosine phosphatase (Griswold-Prenner et al., 1993).
We have described a situation in which the Y333F,Y338F receptor results in activation of JAK2, but with deficient lipogenesis, mitogenesis, and protein synthesis. This may suggest some disjunction between activation of JAK2 and GH stimulation of these events. However, previous studies have demonstrated that cellular tyrosine phosphorylation is required for some of these functions , and a GH receptor with mutations in "Box 1" which is deficient in JAK2 activation is also deficient in these functions (for review see Lobie et al. (1994b)). Thus it is apparent that JAK2 activation (or tyrosine phosphorylation) is necessary but not sufficient for the response to GH. Such a situation also exists for GH stimulation of transcription in which a truncated receptor (at position 455) retains JAK2 activation (VanderKuur et al., 1994) but is unable to fully activate transcription (Goujon et al., 1994). Other signaling molecules which participate in and reconstitute the full cellular effect of GH remain to be identified. One of these is SHC, although this molecule apparently does not associate with the receptor and is not affected by Y333F,Y338F substitution (VanderKuur et al., 1995a).
In conclusion, we have demonstrated that tyrosine residues 333 and 338 of the GH receptor are required for selected cellular responses to GH. We therefore provide the first experimental evidence that tyrosine phosphorylation of the GH receptor is important for GH-stimulated function. In analogy to other receptor signaling systems (Cantley et al., 1991;Nishimura et al., 1993;Mayer and Baltimore, 1993), it is anticipated that these tyrosine residues participate in the recruitment of molecules containing SH2 domains which subsequently execute their cellular effect.