©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A 40-Amino Acid Segment of the Growth Hormone Receptor Cytoplasmic Domain Is Essential for GH-induced Tyrosine-phosphorylated Cytosolic Proteins (*)

(Received for publication, June 27, 1994; and in revised form, November 17, 1994)

Xinzhong Wang Sandra C. Souza(§) (1) Bruce Kelder Joseph A. Cioffi (2) John J. Kopchick (¶)

From the  (1)Department of Biological Sciences, Molecular and Cellular Biological Program and Edison Biotechnology Institute, Ohio University, Athens, Ohio 45701, the Department of Physiology, Medical Center, University of Massachusetts, Worcester, Massachusetts 01655, and (2)Progenitor Inc., Athens, Ohio 45701

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

It has become evident that intracellular protein phosphorylation plays an important role in mediating signal transduction of hormones and growth factors, including growth hormone (GH). We have previously demonstrated that GH can stimulate tyrosine phosphorylation of cellular proteins with approximate molecular masses of 95,000 daltons (pp95) in GH-treated 3T3-F442A preadipocytes and in mouse L cells that express recombinant porcine or bovine GH receptors. In present study, a series of GH receptor (GHR) truncation analogs were constructed and examined for their abilities to induce pp95. The results revealed that a region of 40 amino acids in the porcine GHR cytoplasmic domain is essential for induction of pp95. The results also established that the 115 amino acids(517-638) near the C terminus of porcine GHR are not required for pp95 induction. Moreover, the basal levels of GH-induced pp95 in parental mouse L cells was suppressed by expression of these GHR truncation analogs. This suggests that pp95 induced by GH may be mediated by GHR dimerization and can be inhibited by overexpression of truncated porcine GHRs.


INTRODUCTION

Growth hormone (GH) (^1)exerts its pleiotropic biological functions by first binding with GH receptor(s) on the target cell surface. GH treatment can elicit a variety of responses, such as proto-oncogene induction, enhanced glucose utilization, accumulation of lipid(1, 2, 3, 4) , and activation of protein kinases(5, 6, 7, 8, 9, 10, 11, 12) . GH treatment also promotes conversion of the preadipocytes to adipocytes(13, 14) . However, the signal transduction pathway following the GHbulletGHR interaction remains undefined.

GHR belongs to a superfamily of growth factor and cytokine receptors, which includes prolactin, erythropoietin, interleukins, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, ciliary neurotrophic factor, and leukemia inhibitory factor (15, 16) . The family members share many structural features including a proline-rich sequence in the intracellular portion of the GHR located near the transmembrane region(15) . This proline-rich sequence has been termed ``Box 1''. The Box 1 is hypothesized to be the JAK2 association site and is critical for signal transduction by these receptors(17, 18, 19, 20) . Demonstration of involvement of Janus family kinases (JAK1, JAK2, and Tyk2) in interferon signal transduction pathway(21) , as well as identification of a GHR and erythropoietin receptor associated tyrosine kinase, JAK2, provides further insight into the signaling system of these family members(22, 23) . The possibility that other protein molecules may be involved in the signaling system has not been eliminated.

It has been demonstrated that the GHR itself along with several other cytosolic proteins with molecular masses of 121, 95, 43, and 40 kDa are tyrosine phosphorylated in mouse 3T3-F442A preadipocytes after GH treatment(24, 25) . The 121-kDa protein was identified as the JAK2 tyrosine kinase, and the 43- and 40-kDa proteins have been identified as mitogen-activated protein kinases based upon their co-migration with proteins identified by extracellular signal-regulated kinases 1 and 2 antibodies in GHR cDNA-transfected Chinese hamster ovary cells and 3T3-F442A cells(10, 11, 26) . Recently, interferon-stimulated 91-kDa transcription factor (also known as STAT 1) was found to be phosphorylated on tyrosine residues and shown to bind to the c-sis-inducible element of the c-fos promoter after GH treatment(27) . However, the identity of the 95-kDa proteins has not been reported.

In addition to mouse 3T3-F442A cells, we have previously demonstrated that a cytosolic protein with molecular mass of 95 kDa (pp95) can be induced to be tyrosine phosphorylated by GH treatment in a recombinant MLC which expresses porcine GHR(28) . We have also shown that pp95 phosphorylation is GH-specific in this system. Tyrosine phosphorylation of a protein with similar molecular mass (93 kDa) was also stimulated in a GH-treated human lymphocyte line (IM-9) and was shown to be GH-specific(29) . Therefore, we hypothesized that pp95 or the equivalent protein(s) in other cells may be a mediator for GH signal transduction. Also, it is possible that these molecules may dictate the GH specificity in the GHbulletGHR signal transduction pathway(s).

To determine the relationships between the induction of pp95 after GH treatment and the elements or motifs within the GHR intracellular domain essential for or related to signal transduction, we have selectively generated a series of GHR truncation analogs via site-directed mutagenesis and expressed these mutated cDNAs in MLC. The truncation positions were selected near the 6 conserved tyrosine residues of porcine, bovine, ovine, mouse, rat, and human GHRs. Subsequently, the induction of pp95 by GH treatment was examined in cells that express GHR or these GHR truncation analogs.


MATERIALS AND METHODS

All restriction endonucleases, T4 DNA ligase, T4 DNA polymerase, and Escherichia coli DNA polymerase (Klenow fragment) were obtained from commercial sources (New England Bio-Labs, Berverly, MA). [alpha-P]UTP, [alpha-P]dATP, and I-hGH (100 µCi/µg) were obtained from Dupont NEN. I-pGH (100 µCi/µg) was kindly provided by American Cyanamid Company (Princeton, NJ). Purified pGH was a generous gift from Smith, Kline & Beecham (Westchester, PA). All chemicals used were of reagent grade. The sources of any specific reagents are indicated in the text.

Construction of pGHR cDNAs Which Encode pGHR Truncation Analogs

The full-length pGHR cDNA coding sequence ((30) , EMBL X54429) was inserted into a mammalian expression vector, pMet-IG7, at the XbaI and EcoRI restriction sites. Transcription of pGHR cDNA was directed by the mouse metallothionein I (mMet-I) transcription regulatory sequence and employs the bovine GH (bGH) poly(A) addition signal. The parental plasmid, pMet-IG7, possesses the bacteriophage f1 intergenic region, which enables it to produce single-stranded DNA. Briefly, the 1.9-kilobase DNA fragment, which contains the full-length pGHR cDNA was excised from plasmid pGHR15 (31) by XbaI and EcoRI partial digestion, separated by 1% agarose gel electrophoresis, and purified by Elutip-D column (Schleicher & Schuell). The pGHR cDNA fragment was ligated into XbaI- and EcoRI-digested pMet-IG7. The ligation mixture was transformed into E. coli JM110. Bacterial colonies were grown on LB plates containing 100 µg/ml ampicillin. Colonies containing the pGHR cDNA inserts were screened by restriction digestion analysis. The resulting plasmid containing the pGHR cDNA was termed pMet-IG-pGHR. Single-stranded DNA from pMet-IG-pGHR was prepared by retransforming E. coli CJ236 with pMet-IG-pGHR followed by isolation of the single-stranded DNA, aliquoting, and freezing at -20 °C as described previously(32) .

The pGHR truncation analogs were generated by introducing three translational stop codons via site-directed mutagenesis at the desired positions. Negative strand oligonucleotides were used (National Biosciences, Annapolis, MN). All of the mutagenesis oligonucleotides were 45-50 bases in length with three stop codons (TAGGTAGGTAG) near the middle of the oligonucleotides. The first stop codon in oligonucleotide was used to replace Arg at position 293 for pGHR-TR1(Delta291-638), Ser at 388 for pGHR-TR3 (Delta387-638), Asn at 477 for pGHR-TR4 (Delta476-638), His at 517 for pGHR-TR5, and Ser at 559 for pGHR-TR6. The mutagenesis reactions were carried out as described previously using T4 DNA polymerase(32) . Each reaction mix was used to transform E. coli DH5alpha. Colonies that possess the mutated pGHR cDNA were selected by restriction digestion analysis using XbaI. This XbaI site was engineered into each oligonucleotide following the third stop codon. Each mutation was confirmed by DNA sequencing(33) . The plasmids that contain the mutated pGHR cDNAs are referred to as pMet-IG-pGHR:TR1 through TR6. pGHR-TR2 was generated by deletion of the fragment from the EcoRI site after the transmembrane domain to the EcoRI site located at the 3` end of the intracellular domain, which resulted in deletion of amino acids 337-638 (Delta337-638). pGHR-TR2 utilizes the translational stop codon of bGH. This fusion gene adds 3 amino acids (-Cys-Ala-Phe) derived from the C terminus of bGH to the pGHR-TR2 analog.

Establishment of Stable Cell Lines That Express pGHR Truncation Analogs

pMet-IG-pGHR:TR1, TR2, TR3, TR4, TR5, and TR6 DNAs were used to establish stable MLC lines that permanently express mutated pGHR cDNA. MLC were maintained in Dulbecco's modified Eagle's medium containing 10% Nu-serum I culture supplement (Becton Dickinson), 10 µg/ml gentamicin (Life Technologies, Inc.) at 37 °C in humidified atmosphere containing 5% CO(2). A previously described strategy was employed for transfection(34, 35) . Briefly, MLC were co-transfected with the mutated plasmid DNAs and pRSV-Neo DNA. The transfected cells were cultured with Dulbecco's modified Eagle's medium supplemented with 10% calf serum and G418 (400 µg/ml). G418-resistant colonies were propagated in 25-cm^2 tissue culture flask. Expression of pGHR-TR analogs mRNA was determined either by RNA slot blotting or ribonuclease protection assays. Each cell line was named for the truncation analogs that it expressed, e.g. pGHR-TR1 through pGHR-TR6.

Receptor Binding Analyses of the pGHR-TR Stable Cell Lines

To examine the total GHR binding sites existed in pGHR-TRs cells, maximum displacement experiments (with or without excess amount (2 µg/ml) of unlabeled pGH)) were employed. pGHR-WT cells and MLC were plated in six-well tissue culture plates and propagated to confluence (1 times 10^6 cells). Approximately, 60,000 cpm of I-pGH (100 µCi/µg) was used in each assay. Experiments were performed using methods previously described(31) . Specific binding was calculated as the difference between total binding and nonspecific binding. All determinations were made based upon the mean value of three experiments. Each experiment was carried out in triplicate.

Cross-linking Studies

pGHR-TRs cells, pGHR-WT, and parental MLC (31) were propagated in 25-cm^2 tissue culture flasks. A modification of a previously reported cross-linking procedure was employed(31) . Briefly, the cells were incubated with 100,000 cpm of I-hGH in phosphate-buffered saline, 0.1% bovine serum albumin at room temperature for 2 h and then washed with phosphate-buffered saline, 0.1% bovine serum albumin. pGHR or pGHR-TRs and I-hGH were then cross-linked at room temperature for 60 min with BS^3 (Pierce, Rockford, IN). An excess amount of unlabeled pGH (2 µg/ml) was used in competition reactions. Cells were solubilized in SDS-polyacrylamide gel electrophoresis sample buffer, sonicated for 15 seconds and subjected to 4-12% gradient SDS-polyacrylamide gel electrophoresis(36) . After electrophoresis, the gel was dried and exposed to x-ray film.

pp95 Induction Assay in Cell Lines That Express pGHR Truncation Analogs

To evaluate the biological function of the pGHR truncation analogs in regard to induction of pp95 following GH treatment, pGHR-TRs cells as well as pGHR-WT cells were propagated in six-well tissue culture plates to confluence. GH in the medium was removed by incubating the cells in serum-free medium overnight. The cells were treated with or without 500 ng/ml of pGH at 37 °C for 10 min. Subsequently, the pp95 assay was performed as described previously using the phosphotyrosine antibody, PY20 (ICN, Costa Mesa, CA)(28) .


RESULTS

Construction of pGHR Truncation Analogs

Intracellular pGHR truncation analogs were generated by oligonucleotide directed mutagenesis (see ``Materials and Methods''). Four colonies from each transformation reaction were propagated, and DNA was extracted and sequenced. One clone containing the predicted nucleotide sequence was selected for each GHR truncation. pGHR-TR2 was generated by deletion of the intracellular sequence following the EcoRI sites. This deletion results in a loss of 302 amino acids of the pGHR. In summary, six intracellular domain truncations of pGHR cDNA were generated, and clones were selected: pGHR-TR1 contains pGHR amino acid residues 1-291 or has residues 292-638 deleted (Delta); pGHR-TR2 (1-336 or Delta337-638); pGHR-TR3 (1-387 or Delta388-638); pGHR-TR4 (1-476 or Delta477-638); pGHR-TR5 (1-516 or Delta517-638); and pGHR-TR6 (1-558 or Delta559-638) and are shown schematically (see Fig. 4A).


Figure 4: A, schematic representation of pGHR and pGHR truncation analogs structure. The numbers in parentheses indicate the length of intracellular domain of pGHR or pGHR truncation analogs. The dottedboxes in the pGHR or pGHR analogs indicate the conserved regions among the GH/cytokines receptor family. Also, the pp95 induction results are summarized in lowerpanel as followsbullet filleddot, positive; openeddot, negative. B, alignment of Box 3 amino acid sequences of GHR from various animal species. The positions of pGHR-TR4 and -TR5 are indicated in the sequence via arrows. The shaded amino acids represent the conserved residues among the GHRs. Tyrosine residue at position 487 is indicated by boldface.



Expression of pGHR-TR Analogs in MLC

G418-resistant MLC pools transfected individually with the various pGHR-TR plasmids were grown in 25-cm^2 tissues culture flasks, and total cellular RNA was extracted for expression analyses. Both RNA slot blot and ribonuclease protection analysis revealed that all of the pGHR-TR cell lines express pGHR-TR analogs mRNA at comparable levels with that of the pGHR-WT (data not shown). To further determine the binding capacity of pGHR-TRs to GH, maximum displacement GH binding experiments were performed. The results are summarized in Fig. 1. All of the pGHR-TRs cells were able to bind to I-pGH. The specific binding capacity correlated with RNA expression levels in pGHR-WT and pGHR-TR1 through TR4. However, the binding capacity of pGHR-TR1 and TR2 is much greater than other pGHR-TR analogs (Fig. 1) despite similar levels of pGHR RNA in the cell lines.


Figure 1: Maximum displacement experiments on MLC, pGHR-WT, and cells that express pGHR truncation analogs. Cells were incubated in serum-free medium overnight before the assay was performed. Approximately 60,000 cpm of I-pGH was incubated with the cells in the presence or absence of excess amount of unlabeled pGH at room temperature for 2 h. The cells were washed and harvested. The radioactivity of each sample was determined. Each value represents the results from three experiments (also, see ``Materials and Methods'').



Cross-linking Studies of pGHR Truncation Analogs

To assure that pGHR on the surface of pGHR-TRs cells represents the truncated pGH receptors, cross-linking studies were performed, and results are shown in Fig. 2. In Fig. 2, lanes 1-4 and 9 and 10 represent prolonged exposures (48 h) of the gel. No signal was observed in the parental MLC in either the absence or presence of cold pGH (Fig. 2, lanes 1 and 2). A radiolabeled band with estimated molecular mass of 140 kDa was observed in pGHR-WT cells that could be specifically competed by unlabeled pGH (Fig. 2, lanes3 and 4). This band apparently represents the I-hGHbulletpGHR complex. When the molecular mass of hGH (22 kDa) was subtracted from the complex, the molecular mass of the pGHR was found to be approximately 118 kDa, which agrees with previous reports(31) . Radiolabeled bands were observed in pGHR-TRs cell lines (Fig. 2, lanes5, 7, 9, 11, 13, and 15) and were competed by unlabeled pGH (Fig. 2, lanes6, 8, 10, 12, 14, and 16). The apparent molecular mass of the observed bands in pGHR-TRs cells decreased sequentially as expected for the deletion analogs. The estimated molecular masses of the various pGHR-TR analogs were as follows: pGHR-TR6, 126 kDa; pGHR-TR5, 120 kDa; pGHR-TR4, 117 kDa; pGHR-TR3, 98 kDa; pGHR-TR2, 81 kDa; and pGHR-TR1, 74 kDa. Subtraction of the molecular mass of hGH from these complexes results in pGHR-TR1, TR2, TR3, TR4, TR5, and TR6 possessing approximate molecular masses of 52, 59, 76, 95, 98, and 104 kDa, respectively.


Figure 2: Autoradiography of GH-GHR cross-linking experiments on MLC and MLC lines that express full-length pGHR or its truncation analogs. - and + indicate the cross-linking reactions were performed in the absence or presence of excess amount (2 µg/ml) of unlabeled pGH. Lanes1-4, 9 and 10 are prolonged exposures of the same gel.



Moreover, a second band was observed in the cross-linking studies of pGHR-TR1 and TR2 (Fig. 2, lanes15 and 13) and less distinct bands were seen in lanes3, 5, 7, 9, and 11 of Fig. 2. The molecular masses of these bands decreased, concomitantly, with the size of GHbulletGHR monomer and could be competed by cold pGH specifically. The estimated molecular masses of these bands are 156 ± 6 and 163 ± 6 kDa for pGHR-TR1 and pGHR-TR2, respectively (summary of results from five cross-linking gels).

pp95 Induction Assay in pGHR-TRs Cells

To examine the effects of truncations of pGHR intracellular domain in GHbulletGHR signal transduction, the ability of GH to induce tyrosine-phosphorylated proteins with a molecular mass of 95,000 daltons (pp95) was examined. The results were shown in Fig. 3. As described previously, basal levels of pp95 induction were observed in GH-treated parental MLC (Fig. 3, lane2). GH treatment induced pp95 in cells that express pGHR-WT (28) (Fig. 3, lane4). The pp95 signal was induced to a similar level in pGHR-TR5 and TR6 cells. No pp95 induction was observed in pGHR-TR1, TR2, TR3, and TR4 cells after GH treatment. These results are summarized in Fig. 4A.


Figure 3: pp95 induction assay on cell lines that express full-length GHR or its truncation analogs. Cells were plated in six-well tissue culture plates. GH in the medium was removed by incubating the cells in serum-free medium overnight. Subsequently, cells were treated with or without pGH (500 ng/ml) for 10 min at 37 °C and processed as described in ``Materials and Methods.'' The arrow on left indicates the position of pp95.



Interestingly, in addition to the lack of pp95 induction in pGHR-TR1, TR2, and TR3 cells, the basal levels of pp95 induction was also diminished (Fig. 3).


DISCUSSION

GHR Intracellular Domain Structure and pp95 Induction

Employing a cDNA mutagenesis/protein truncation approach for the intracellular region of the pGHR, our results reveal a region of 40 amino acids (from 477 to 516) that is essential for GH induction of pp95. Alignment of the amino acid sequence of the 40-amino acid segment of pGHR with the corresponding regions of GHR from other species shows a relatively high degree of amino acid identity (Fig. 4B). Importantly, this 40-amino acid domain is distinct from the ``proline-rich'' region or Box 1 described previously among all the GHRs as well as prolactin and other cytokine receptors(17, 18, 19, 20) . It has been demonstrated that the proline-rich region plays a critical role in mediating GH signal transduction(19, 20) .

A tyrosine residue (Tyr-487) is conserved among the GHRs from all species in the identified 40-amino acid segment (Fig. 4B). We propose that Tyr-487 of pGHR or the corresponding tyrosine residue of GHRs from other species may be important for the induction of pp95 tyrosine phosphorylation. We hypothesize that Tyr-487 is phosphorylated by the GHR-associated tyrosine kinase, JAK2(22) . The phosphorylated Tyr-487 would provide a docking site for proteins that possess Src-homology (SH2) domains and may play important roles in GH signal transduction as described for the epidermal growth factor receptor or insulin receptors(37) .

In addition to the proline-rich region, it has been shown that the 80 amino acids at the C-terminal region of GHR are required for activation of serine protease inhibitor promoter(38) . Also, it has been shown that deletion of the C terminus of GHR results in loss of stimulation of insulin production in insulinoma RIN-5AH cells(39) . Our data demonstrate that the carboxyl-terminal 115 amino acids(517-638) are not required for pp95 induction. Combining the present and previously described data, it is reasonable to deduce that multiple motifs within the GHR intracellular domain may mediate the pleiotropic biological functions of GH. Thus, elucidation of GHbulletGHR interaction, as well as the linkage between the GHR (especially the cytoplasmic domain of the GHR), and the intracellular elements becomes very important in understanding GHbulletGHR signal transduction.

No pp95 induction was observed in pGHR-TR1 cells, i.e. cells that lack the intracellular domain. This result agrees with others that found that the cytoplasmic domain is essential for GH signal transduction(18, 19) . However, pGHR-TR2, TR3, and TR4 possess the intact Box 1 sequence and, presumably, provide sufficient elements for JAK2 kinase association but still lack the ability to induce pp95. This could be explained in the following two ways. 1) pp95 is not phosphorylated by the GHR-associated tyrosine kinase, JAK2, which would imply that pp95 and JAK2 kinase belong to different pathways of GH signal transduction. 2) pp95 phosphorylation is dependent on JAK2 activity. If the latter possibility is true, then truncation of the GHR intracellular domain or the 40-amino acid box 3 sequence would result in loss of a putative pp95 recruiting site(s) and would not lead to phosphorylation by JAK2. In this scenario, the pp95 protein requires access to the GHR (e.g. association with the 40-amino acid segment directly or through other anchor proteins) to be phosphorylated by the JAK2 kinase.

Interestingly, basal levels of pp95 induction were not observed in pGHR-TR1, TR2, and TR3 cells. We assume this phenomenon, a ``dominant negative effect,'' is caused by the overexpression of nonfunctional pGHR-TRs in MLC. Overexpressed pGHR-TRs may lead to the formation of a pGHR-TRbulletGHbulletmGHR heterodimer, which results in suppression of basal levels pp95 induction in MLC. These heterodimers could not be observed on the cross-linking gel probably because of the extremely low levels of mGHR in MLC (Fig. 2, lane1)(31) . The formation of pGHR-TRbulletGHbulletmGHR heterodimers would also not transduce the GH signal. Together with the observations of pGHRbulletGHbulletpGHR formation, we propose that the induction is not only dependent on a functional motif(s) in the cytoplasmic domain but also on the dimerization of the GHRs.

GH and GHR Complex Dimerization and Internalization

In the present study, we used an in vitro mutagenesis approach to generate pGHR cytoplasmic domain truncation analogs. Transfection of MLC with these mutated pGHR cDNAs resulted in at least a 10-100 fold increase of I-pGH binding at the cell surface (Fig. 1). The truncations of the pGHR cytoplasmic domain did not affect the binding ability of pGHR to pGH. We have previously shown that the binding affinity of pGHR-TR1, which possesses the shortest (3 amino acids) intracellular portion of pGHR, was found to be similar to wild-type pGHR, i.e. 1.0 times 10M (data not shown). All other pGHR truncations, which possess longer intracellular domains than pGHR-TR1, did not change GH binding affinity.

Cross-linking studies demonstrated that the pGH-TRs produced were of correct apparent molecular masses. Furthermore, in the cell lines that expressed pGHR analogs, pGHR-TR1 and TR2, a second band in addition to the band that represents the GHbulletGHR monomer was observed (Fig. 2, lanes13 and 15). However, based on their calculated molecular masses, the size of these bands do not correspond to the GHRbulletGHbulletGHR complex as described previously(40) . We have performed statistical analysis of the data collected from five different cross-linking gels. For example, the molecular mass of pGHR-TR1 is 52 ± 5 kDa (n = 5). The molecular mass of pGHR-TR1bulletpGHbulletpGHR-TR1 complex should be 126 ± 10 kDa. However, the molecular mass of the second band observed in pGHR-TR1 cells is 156 ± 6 kDa (n = 5). Therefore, the second band may represent a different form of GHbulletGHR complex than a GHRbulletGHbulletGHR complex. We hypothesize that these bands may represent three possible GHbulletGHR complexes. 1) The first is a GHRbulletGHbulletGHbulletGHR complex since the difference between the calculated molecular mass and the observed molecular mass is 30 kDa for TR1 and 22 kDa for TR2. Therefore, these differences might indicate a second GH molecule in the GHRbulletGHbulletGHR complex. 2) The second is a GHRbulletGHbulletGHRbulletX complex, whereas X represents an unknown protein molecule with a molecular mass of 25 kDa associated with the GHRbulletGHbulletGHR complex. 3) And finally is a GHRbulletGHbulletY complex, where Y represents an unknown protein molecule with a molecular mass of 104 kDa (a GH signal mediator) similar the gp130 molecule used in interleukin 6 signal transduction(41) . This difference in expected size obtained here relative to the published report may be due to a difference in experimental systems. The GHBPbulletGHbulletGHBP complex model was established by use of E. coli-produced GH binding protein, which is not glycosylated or membrane-bound(40) . Our results were obtained from intact mammalian cells that express the glycosylated, membrane-bound form of the GHR analogs. It has been proposed that the use of living cells producing GHR rather than bacterially produced GH binding protein may generate more realistic results related to the GHbulletGHR interaction(42) . However, it is possible that various forms of GHbulletGHR dimer exist and mediate different GH signals. More detailed studies are required to test these hypothetical models. Regardless of the GHbulletGHR complex model, it was shown that the truncations of pGHR cytoplasmic domain do not affect the ability of GHR to form GHbulletGHR complexes. The absence of the GHbulletGHR dimer complex in pGHR-TR6, TR5, TR4, and TR3 cells was probably due to fewer GH binding sites on these cells.

By comparing the expression levels of pGHR-TRs in the pGHR-TRs cell lines to the pGHR binding sites, it was found that the difference in expression levels was not as variable as the difference in the number of binding sites. This may result from the fact that TR1 and TR2 lack the GHR internalization signals that, therefore, lead to accumulation of pGHR-TRs on the cell surface. Receptor internalization assays revealed the internalization rates of pGHR-TR1 and pGHR-TR2 were 60% lower than that of the wild-type pGHR in MLC. (^2)Also, this explanation is supported by the results reported by Möldrup and colleagues (39) in which a GHR containing a deletion of the majority of the intracellular domain (GHR) was found to have a dramatically decreased internalization rate compared with the wild-type GHR. In the same study, another GHR deletion mutant (GHR) retains the same internalization rate. The GHR is similar to our pGHR-TR4 (1-476 or Delta477-638). Therefore, our data suggests that the ``internalization signal'' of GHRs is located between amino acid 337 and 387 (from pGHR-TR2 to TR3, 50 amino acids), which is adjacent to the proline-rich region.

In conclusion, we have found a 40-amino acid region of the pGHR cytoplasmic domain(477-516), which is essential for the induction of pp95 tyrosine phosphorylation in cells that express pGHR following GH treatment. The results suggest that the pp95 induction by GH is not only dependent on the existence of this 40-amino acid segment in the GHR cytoplasmic domain, but it also requires the formation of GHbulletGHR complex that may be a receptor dimer or a yet to be defined complex. Following the terminology of Box 1 and Box 2 for other members of the cytokine receptor superfamily(19, 20) , we refer to this 40-amino acid region as Box 3 (Fig. 4B).


FOOTNOTES

*
This work was supported in part by the state of Ohio's Eminent Scholar Program, which includes a grant from Milton and Lawrence Goll (to J. K. K.). This work was also partially supported by grants from National Research Initiative Competitive Grants Program/United States Department of Agriculture(91-37206-6738) and National Institutes of Health Grant (DK 42137-01A2) as well as a grant from Innovations in Drug Development, Inc. 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X54429[GenBank].

§
Partially supported by Conselho Nacional de Pequisas, Brazil, Process 201270/90.0.

To whom correspondence should be addressed: Edison Biotechnology Inst., Wilson Hall/West Green, Ohio University, Athens, OH 45701. Tel.: 614-593-4713; Fax: 614-593-4795.

(^1)
The abbreviations used are: GH, growth hormone; GHR, GH receptor; MLC, mouse L cell; hGH, human GH; pGH, porcine GH; bGH, bovine GH.

(^2)
X. Wang, S. C. Souza, and J. J. Kopchick, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. Paul Harding for the valuable suggestions and discussion about the results of present work.


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