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Growth Hormone Receptor Is a Target for Presenilin-dependent γ-Secretase Cleavage

2005; Elsevier BV; Volume: 280; Issue: 19 Linguagem: Inglês

10.1074/jbc.m500621200

ISSN

1083-351X

Autores

Jon W. Cowan, Xiangdong Wang, Ran Guan, Kai He, Jing Jiang, Gerhard Baumann, Roy A. Black, Michael S. Wolfe, Stuart J. Frank,

Tópico(s)

Metabolism, Diabetes, and Cancer

Resumo

Growth hormone receptor (GHR) is a cytokine receptor superfamily member that binds growth hormone (GH) via its extracellular domain and signals via interaction of its cytoplasmic domain with JAK2 and other signaling molecules. GHR is a target for inducible metalloprotease-mediated cleavage in its perimembranous extracellular domain, a process that liberates the extracellular domain as the soluble GH-binding protein and leaves behind a cell-associated GHR remnant protein containing the transmembrane and cytoplasmic domains. GHR metalloproteolysis can be catalyzed by tumor necrosis factor-α-converting enzyme (ADAM-17) and is associated with down-modulation of GH signaling. We now study the fate of the GHR remnant protein. By anti-GHR cytoplasmic domain immunoblotting, we observed that the remnant induced in response to phorbol ester or platelet-derived growth factor has a reliable pattern of appearance and disappearance in both mouse preadipocytes endogenously expressing GHR and transfected fibroblasts expressing rabbit GHR. Lactacystin, a specific proteasome inhibitor, did not appreciably change the time course of remnant appearance or clearance but allowed detection of the GHR stub, a receptor fragment slightly smaller than the remnant but containing the C terminus of the remnant (receptor cytoplasmic domain). In contrast, MG132, another (less specific) proteasome inhibitor, strongly inhibited remnant clearance and prevented stub appearance. Inhibitors of γ-secretase, an aspartyl protease, also prevented the appearance of the stub, even in the presence of lactacystin, and concomitantly inhibited remnant clearance in the same fashion as MG132. In addition, mouse embryonic fibroblasts derived from presenilin 1 and 2 (PS1/2) knockouts recapitulated the γ-secretase inhibitor studies, as compared with their littermate controls (PS1/2 wild type). Confocal microscopy indicated that the GHR cytoplasmic domain became localized to the nucleus in a fashion dependent on PS1/2 activity. These data indicate that the GHR is subject to sequential proteolysis by metalloprotease and γ-secretase activities and may suggest GH-independent roles for the GHR. Growth hormone receptor (GHR) is a cytokine receptor superfamily member that binds growth hormone (GH) via its extracellular domain and signals via interaction of its cytoplasmic domain with JAK2 and other signaling molecules. GHR is a target for inducible metalloprotease-mediated cleavage in its perimembranous extracellular domain, a process that liberates the extracellular domain as the soluble GH-binding protein and leaves behind a cell-associated GHR remnant protein containing the transmembrane and cytoplasmic domains. GHR metalloproteolysis can be catalyzed by tumor necrosis factor-α-converting enzyme (ADAM-17) and is associated with down-modulation of GH signaling. We now study the fate of the GHR remnant protein. By anti-GHR cytoplasmic domain immunoblotting, we observed that the remnant induced in response to phorbol ester or platelet-derived growth factor has a reliable pattern of appearance and disappearance in both mouse preadipocytes endogenously expressing GHR and transfected fibroblasts expressing rabbit GHR. Lactacystin, a specific proteasome inhibitor, did not appreciably change the time course of remnant appearance or clearance but allowed detection of the GHR stub, a receptor fragment slightly smaller than the remnant but containing the C terminus of the remnant (receptor cytoplasmic domain). In contrast, MG132, another (less specific) proteasome inhibitor, strongly inhibited remnant clearance and prevented stub appearance. Inhibitors of γ-secretase, an aspartyl protease, also prevented the appearance of the stub, even in the presence of lactacystin, and concomitantly inhibited remnant clearance in the same fashion as MG132. In addition, mouse embryonic fibroblasts derived from presenilin 1 and 2 (PS1/2) knockouts recapitulated the γ-secretase inhibitor studies, as compared with their littermate controls (PS1/2 wild type). Confocal microscopy indicated that the GHR cytoplasmic domain became localized to the nucleus in a fashion dependent on PS1/2 activity. These data indicate that the GHR is subject to sequential proteolysis by metalloprotease and γ-secretase activities and may suggest GH-independent roles for the GHR. As evidenced by the phenotypes of naturally occurring mutations in humans and targeted gene disruption in mice, the growth hormone receptor (GHR) 1The abbreviations used are: GH, growth hormone; GHR, GH receptor; GHBP, GH-binding protein; rbGHR, rabbit GHR; PDGF, platelet-derived growth factor; PS1/2, presenilin 1 and 2; WT, wild type; STAT, signal transducers and activators of transcription; PMA, phorbol 12-myristate 13-acetate; APP, amyloid precursor protein; IC3, Immunex Compound 3; DMEM; Dulbecco's modified Eagle's medium; HA, hemagglutinin; PBS, phosphate-buffered saline; MEF, mouse embryo fibro-blast; PIPES, 1,4-piperazinediethanesulfonic acid; GSI, γ-secretase inhibitor(s).1The abbreviations used are: GH, growth hormone; GHR, GH receptor; GHBP, GH-binding protein; rbGHR, rabbit GHR; PDGF, platelet-derived growth factor; PS1/2, presenilin 1 and 2; WT, wild type; STAT, signal transducers and activators of transcription; PMA, phorbol 12-myristate 13-acetate; APP, amyloid precursor protein; IC3, Immunex Compound 3; DMEM; Dulbecco's modified Eagle's medium; HA, hemagglutinin; PBS, phosphate-buffered saline; MEF, mouse embryo fibro-blast; PIPES, 1,4-piperazinediethanesulfonic acid; GSI, γ-secretase inhibitor(s). is essential for mediation of the profound metabolic and somatogenic effects of GH (1Laron Z. J. Clin. Endocrinol. Metab. 1995; 80: 1426-1531Google Scholar, 2Parks J.S. Brown M.R. Faase M.E. J. Pediatr. 1997; 131: S45-S50Abstract Full Text Full Text PDF PubMed Google Scholar, 3Zhou Y. Xu B.C. Maheshwari H.G. He L. Reed M. Lozykowski M. Okada S. Cataldo L. Coschigamo K. Wagner T.E. Baumann G. Kopchick J.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13215-13220Crossref PubMed Scopus (654) Google Scholar). GHR is a single membrane-spanning cell surface type 1 glycoprotein member of the cytokine receptor superfamily (4Frank S.J. Messina J.L. Oppenheim J.J. Feldman M. Cytokine Reference On-Line. Academic Press, Harcourt, London, UK2002Google Scholar). It transduces signals in response to GH by coupling to activation of the STAT (1, 3, and 5b), mitogen-activated protein kinase (extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38), and phosphatidylinositol 3-kinase pathways to affect the expression of various target genes, such as insulin-like growth factor-1, serine protease inhibitor 2.1 (Spi2.1), and c-fos, and other aspects of cellular function (4Frank S.J. Messina J.L. Oppenheim J.J. Feldman M. Cytokine Reference On-Line. Academic Press, Harcourt, London, UK2002Google Scholar, 5Carter Su C. Schwartz J. Smit L.S. Annu. Rev. Physiol. 1996; 58: 187-207Crossref PubMed Scopus (273) Google Scholar). Essential to this coupling is the GH-induced activation of the cytoplasmic tyrosine kinase, JAK2, which associates with the cytoplasmic domain of the receptor (6Argetsinger L.S. Campbell G.S. Yang X. Witthuhn B.A. Silvennoinen O. Ihle J.N. Carter-Su C. 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Treatment of a variety of cell types with the phorbol ester, PMA, or platelet-derived growth factor (PDGF) results in loss of the full-length receptor and appearance of a cell-associated cytoplasmic domain-containing GHR fragment that we have termed the “remnant” protein (12Goldsmith J.F. Lee S.J. Jiang J. Frank S.J. Am. J. Physiol. 1997; 273: E932-E941PubMed Google Scholar, 13Alele J. Jiang J. Goldsmith J.F. Yang X. Maheshwari H.G. Black R.A. Baumann G. Frank S.J. Endocrinology. 1998; 139: 1927-1935Crossref PubMed Google Scholar, 14Guan R. Zhang Y. Jiang J. Baumann C.A. Black R.A. Baumann G. Frank S.J. Endocrinology. 2001; 142: 1137-1147Crossref PubMed Scopus (41) Google Scholar, 15Zhang Y. Guan R. Jiang J. Kopchick J.J. Black R.A. Baumann G. Frank S.J. J. Biol. Chem. 2001; 276: 24565-24573Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). This cleavage also yields a soluble GHR form comprised of the extracellular domain of the receptor, which is referred to as the GH-binding protein (GHBP), in correspondence with the high affinity GH-binding protein found in the circulation of many species (16Baumann G. J. Pediatr. Endocrinol. Metab. 2001; 14: 355-375Crossref PubMed Scopus (118) Google Scholar). In humans and rabbits (and likely in many other species), this process of proteolytic shedding of the GHR extracellular domain accounts for the generation of GHBP in vivo (16Baumann G. J. Pediatr. Endocrinol. Metab. 2001; 14: 355-375Crossref PubMed Scopus (118) Google Scholar). By use of hydroxamate-based inhibitors and genetic reconstitution strategies, we have previously implicated metalloprotease activity as required for inducible GHR proteolysis in cell culture systems and have identified a particular transmembrane metalloprotease, tumor necrosis factor-α-converting enzyme (ADAM-17), as a GHBP sheddase (13Alele J. Jiang J. Goldsmith J.F. Yang X. Maheshwari H.G. Black R.A. Baumann G. Frank S.J. Endocrinology. 1998; 139: 1927-1935Crossref PubMed Google Scholar, 14Guan R. Zhang Y. Jiang J. Baumann C.A. Black R.A. Baumann G. Frank S.J. Endocrinology. 2001; 142: 1137-1147Crossref PubMed Scopus (41) Google Scholar, 17Zhang Y. Jiang J. Black R.A. Baumann G. Frank S.J. Endocrinology. 2000; 141: 4324-4348Google Scholar). Our work has also indicated that metalloproteolytic GHR processing may be a mechanism of regulation of cellular GH sensitivity, in that GH-induced signaling is dampened after exposure to stimuli that promote receptor cleavage, but not if metalloprotease inhibitors are present or if noncleavable receptor mutants are expressed (14Guan R. Zhang Y. Jiang J. Baumann C.A. Black R.A. Baumann G. Frank S.J. Endocrinology. 2001; 142: 1137-1147Crossref PubMed Scopus (41) Google Scholar, 18Wang X. He K. Gerhart M. Huang Y. Jiang J. Paxton R.J. Yang S. Lu C. Menon R.K. Black R.A. Baumann G. Frank S.J. J. Biol. Chem. 2002; 277: 50510-50519Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). We recently mapped the metalloprotease-mediated cleavage sites in the rabbit (rb) and mouse GHRs at eight and nine residues, respectively, from the transmembrane domain in the receptor extracellular domain stalk region (18Wang X. He K. Gerhart M. Huang Y. Jiang J. Paxton R.J. Yang S. Lu C. Menon R.K. Black R.A. Baumann G. Frank S.J. J. Biol. Chem. 2002; 277: 50510-50519Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 19Wang X. He K. Gerhart M. Jiang J. Paxton R.L. Menon R.K. Black R.A. Baumann G. Frank S.J. Mol. Endocrinol. 2003; 17: 1931-1943Crossref PubMed Scopus (16) Google Scholar). This was accomplished by purifying the remnant protein and performing N-terminal amino acid sequencing, confirming that the remnant contains the transmembrane domain and the remaining 8–9 (depending on species) extracellular domain residues, as well as the cytoplasmic domain. 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Formation of an active γ-secretase complex depends upon four proteins: presenilin, APH-1, nicastrin (APH-2), and Pen-2 (32Wolfe M.S. Kopan R. Science. 2004; 305: 1119-1123Crossref PubMed Scopus (307) Google Scholar). Coexpression of these proteins is sufficient for reconstitution of γ-secretase activity in yeast, which not only lacks this protease activity but also has no apparent orthologs of these proteins (33Edbauer D. Winkler E. Regula J.T. Pesold B. Steiner H. Haass C. Nat. Cell. Biol. 2003; 5: 486-488Crossref PubMed Scopus (767) Google Scholar). Of these four proteins, it is believed that presenilin itself forms the catalytic core by functioning as an aspartyl protease (32Wolfe M.S. Kopan R. Science. 2004; 305: 1119-1123Crossref PubMed Scopus (307) Google Scholar). γ-Secretase-mediated cleavage of proteins creates soluble intracellular domains that have signaling functions; thus, this process, referred to as regulated intramembrane proteolysis, has attracted increasing interest as an important mechanism for modulating cellular responsiveness. In this report, we explored the fate of the metalloprotease-generated GHR remnant protein. Using both endogenous GHR-expressing cells and GHR reconstitution systems, we demonstrate that the remnant has a characteristic time course of degradation. Our results indicate that the remnant undergoes transition into a slightly smaller form (the “stub”; see Fig. 9) that is itself degraded in a proteasome activity-dependent fashion. This remnant-to-stub transition is inhibited by γ-secretase inhibitors and does not occur in cells devoid of presenilins, suggesting that the GHR remnant is a direct substrate for presenilin-dependent γ-secretase cleavage. Further, in the presence of proteasome inhibitors, we detect γ-secretase-dependent accumulation of the GHR cytoplasmic domain in the nucleus. The parallels revealed by our studies between the GHR and other γ-secretase substrates, such as APP and Notch, suggest that a functional role(s) for metalloprotease/γ-secretase cleavage of GHR may exist. Materials—PMA and routine reagents were purchased from Sigma unless otherwise noted. MG132 (Z-LLL-CHO) was purchased from Calbiochem (La Jolla, CA). Immunex Compound 3 (IC3), supplied by Amgen Corporation, is identical to Compound 2 (34Mohler K.M. Sleath P.R. Fitzner J.N. Cerretti D.P. Alderson M. Kerwar S.S. Torrance D.S. Otten-Evans C. Greenstreet T. Weerawarna K. et al.Nature. 1994; 370: 218-220Crossref PubMed Scopus (570) Google Scholar), 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. The γ-secretase inhibitors used for each experiment are noted in the figure legends. The IC50 of Compound E is 0.3 nm (35Seiffert D. Bradley J.D. Rominger C.M. Rominger D.H. Yang F. Meredith Jr., J.E. Wang Q. Roach A.H. Thompson L.A. Spitz S.M. Higaki J.N. Prakash S.R. Combs A.P. Copeland R.A. Arneric S.P. Hartig P.R. Robertson D.W. Cordell B. Stern A.M. Olson R.E. Zaczek R. J. Biol. Chem. 2000; 275: 34086-34091Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar), and the IC50 for N-[N-(3,5-difluorophenacetyl-l-alanyl]-S-phenylglycine-t-butyl ester (DAPT) is 20 nm (36Dovey H.F. John V. Anderson J.P. Chen L.Z. de Saint Andrieu P. Fang L.Y. Freedman S.B. Folmer B. Goldbach E. Holsztynska E.J. Hu K.L. Johnson-Wood K.L. Kennedy S.L. Kholodenko D. Knops J.E. Latimer L.H. Lee M. Liao Z. Lieberburg I.M. Motter R.N. Mutter L.C. Nietz J. Quinn K.P. Sacchi K.L. Seubert P.A. Shopp G.M. Thorsett E.D. Tung J.S. Wu J. Yang S. Yin C.T. Schenk D.B. May P.C. Altstiel L.D. Bender M.H. Boggs L.N. Britton T.C. Clemens J.C. Czilli D.L. Dieckman-McGinty D.K. Droste J.J. Fuson K.S. Gitter B.D. Hyslop P.A. Johnstone E.M. Li W.Y. Little S.P. Mabry T.E. Miller F.D. Audia J.E. J. Neurochem. 2001; 76: 173-181Crossref PubMed Scopus (787) Google Scholar). These values were derived from cell-based APP γ-secretase cleavage assays and are used as guidelines. The actual IC50 values as related to the γ-secretase cleavage of the GHR have not been determined. Cells, Cell Culture, Transfection, and Adenoviral Infection—HEK-293 human embryonic kidney cells were maintained in DMEM (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). Adenoviral infection and transient transfection of HEK-293 cells was accomplished using methods previously reported (18Wang X. He K. Gerhart M. Huang Y. Jiang J. Paxton R.J. Yang S. Lu C. Menon R.K. Black R.A. Baumann G. Frank S.J. J. Biol. Chem. 2002; 277: 50510-50519Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 37Lu C. Schwartzbauer G. Sperling M.A. Devaskar S.U. Thamotharan S. Robbins P.D. McTiernan C.F. Liu J.L. Jiang J. Frank S.J. Menon R.K. J. Biol. Chem. 2001; 276: 22892-22900Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). 3T3-F442A murine preadipocyte cells (38Green H. Kehinde O. Cell. 1976; 7: 105-113Abstract Full Text PDF PubMed Scopus (611) Google Scholar), kindly provided by Drs. H. Green (Harvard University) and C. Carter-Su (University of Michigan), were maintained in DMEM (4.5 g/liter glucose) supplemented with 10% calf serum (Biofluids) and 50 μg/ml gentamicin sulfate, 100 units/ml penicillin, and 100 μg/ml streptomycin (all Biofluids). γ2A-GHR cells, which are JAK2-deficient human fibrosarcoma cells that express the rabbit GHR (15Zhang Y. Guan R. Jiang J. Kopchick J.J. Black R.A. Baumann G. Frank S.J. J. Biol. Chem. 2001; 276: 24565-24573Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), were maintained in DMEM (1 g/liter glucose) supplemented with 10% fetal bovine serum (Biofluids) and 50 μg/ml gentamicin sulfate, 100 units/ml penicillin, and 100 μg/ml streptomycin (all Biofluids) and 200 μg/ml of both G418 and hygromycin (Invitrogen). PS1/2 knock-out cells, murine embryonic fibroblasts with targeted deletion of presenilins 1 and 2 (39Herreman A. Serneels L. Annaert W. Collen D. Schoonjans L. De Strooper B. Nat. Cell Biol. 2000; 2: 461-462Crossref PubMed Scopus (448) Google Scholar), kindly provided by Dr. B. De Strooper (K.U. Leuven, Belgium), were maintained in the same medium as γ2A-GHR cells. PS1/2 WT cells, littermate control murine embryonic fibroblasts (39Herreman A. Serneels L. Annaert W. Collen D. Schoonjans L. De Strooper B. Nat. Cell Biol. 2000; 2: 461-462Crossref PubMed Scopus (448) Google Scholar), were maintained in identical medium lacking G418 and hygromycin. γ2A-JAK2 cells, γ2A stably reconstituted with murine JAK2 (40Jiang J. Wang X. He K. Li X. Chen C. Sayeski P.P. Waters M.J. Frank S.J. Mol. Endocrinol. 2004; 18: 2981-2996Crossref PubMed Scopus (43) Google Scholar, 41Wallace T.M. Van Der Linden D. He K. Frank S.J. Sayeski P.P. Am. J. Physiol. Cell Physiol. 2004; 287: C981-C991Crossref PubMed Scopus (14) Google Scholar), kindly provided by Dr. P. P. Sayeski (University of Florida), were maintained in medium identical to γ2A-GHR; however, 5 μg/ml Zeocin (Invitrogen) was used to replace hygromycin. R+T mouse fibroblasts (17Zhang Y. Jiang J. Black R.A. Baumann G. Frank S.J. Endocrinology. 2000; 141: 4324-4348Google Scholar) were maintained in 50/50 Ham's F-12/DMEM (low glucose) supplemented with 5% fetal bovine serum (Biofluids) and 50 μg/ml gentamicin sulfate, 100 units/ml penicillin, and 100 μg/ml streptomycin (all Biofluids), 15 mm HEPES, pH 7.5, and 200 μg/ml hygromycin B (Mediatech, Inc., Herndon, VA). Plasmid Construction—The rbGHR cDNA was a kind gift of Dr. W. Wood (Genentech, Inc.). Construction of the cDNA encoding rbGHRdel 297–406-His has been described (7Frank S.J. Gilliland G. Kraft A.S. Arnold C.S. Endocrinology. 1994; 135: 2228-2239Crossref PubMed Scopus (107) Google Scholar, 18Wang X. He K. Gerhart M. Huang Y. Jiang J. Paxton R.J. Yang S. Lu C. Menon R.K. Black R.A. Baumann G. Frank S.J. J. Biol. Chem. 2002; 277: 50510-50519Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 19Wang X. He K. Gerhart M. Jiang J. Paxton R.L. Menon R.K. Black R.A. Baumann G. Frank S.J. Mol. Endocrinol. 2003; 17: 1931-1943Crossref PubMed Scopus (16) Google Scholar, 42Zhang Y. Jiang J. Kopchick J.J. Frank S.J. J. Biol. Chem. 1999; 274: 33072-33084Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). This mutant has intact extracellular and transmembrane domains but lacks residues 297–406 in the cytoplasmic domain (the full-length rbGHR has 620 residues). 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 (43Govers R. ten Broeke T. van Kerkhof P. Schwartz A.L. Strous G.J. EMBO J. 1999; 18: 28-36Crossref PubMed Scopus (169) Google Scholar, 44Allevato G. Billestrup N. Goujon L. Galsgaard E.D. Norstedt G. Postel-Vinay M.C. Kelly P.A. Nielsen J.H. J. Biol. Chem. 1995; 270: 17210-17214Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) is absent. The rbGHRdel 297–406 del 237–239-His mutant has also been described (18Wang X. He K. Gerhart M. Huang Y. Jiang J. Paxton R.J. Yang S. Lu C. Menon R.K. Black R.A. Baumann G. Frank S.J. J. Biol. Chem. 2002; 277: 50510-50519Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In addition to internal deletion of residues 297–406, this mutant also lacks residues 237–239, which includes the rb-GHR cleavage site, and is thus resistant to metalloprotease-mediated proteolysis (18Wang X. He K. Gerhart M. Huang Y. Jiang J. Paxton R.J. Yang S. Lu C. Menon R.K. Black R.A. Baumann G. Frank S.J. J. Biol. Chem. 2002; 277: 50510-50519Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The pcDNA 3.1(-) rbGHR239–620-HA recombinant remnant corresponds to the mapped metalloprotease cleavage site but contains both the GHR signal sequence and a C-terminal HA tag following spacer residues. cDNAs encoding both rbGHRdel 297–406-His and rbGHRdel 297–406 del237–239-His were in the pcDNA 3.1(-) eukaryotic expression vector, as previously reported. Generation of Recombinant Adenoviruses—The methods for generating the adenovirally expressed version of rbGHRdel 297–406-His were described previously (37Lu C. Schwartzbauer G. Sperling M.A. Devaskar S.U. Thamotharan S. Robbins P.D. McTiernan C.F. Liu J.L. Jiang J. Frank S.J. Menon R.K. J. Biol. Chem. 2001; 276: 22892-22900Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Briefly, rbGHRdel297–406-His cDNA was removed from pcDNA3.1(-) rbGHRdel297–406-His by XbaI and KpnI digestion and inserted into pAdlox vector to form pAdlox-rbGHRdel-297–406-His. Linearized pAdlox-rbGHRdel 297–406-His and Ψ5 helper virus DNA were cotransfected into CRE8 cells (45Hardy S. Kitamura M. Harris-Stansil T. Dai Y. Phipps M.L. J. Virol. 1997; 71: 1842-1849Crossref PubMed Google Scholar) (an HEK-293 derivative) by Lipofectamine (Invitrogen). The cells were harvested after several days when the cytopathic effects became apparent. After lysis by three freeze/thaw cycles, the cell debris was pelleted by centrifugation, and the 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. WT rbGHR-His adenovirus was constructed in the same way as Ad rbGHRdel 297–406-His. Antibodies—The 3F10 anti-HA rat monoclonal antibody was purchased from Roche. Three rabbit polyclonal antisera against the GHR were used. Anti-GHRcyt-AL47 was raised against a bacterially expressed N-terminally His-tagged fusion protein incorporating human GHR residues 271–620 (the entire cytoplasmic domain (46Leung D.W. Spencer S.A. Cachianes G. Hammonds R.G. Collins C. Henzel W.J. Barnard R. Waters M.J. Wood W.I. Nature. 1987; 330: 537-543Crossref PubMed Scopus (1318) Google Scholar)), as described (15Zhang Y. Guan R. Jiang J. Kopchick J.J. Black R.A. Baumann G. Frank S.J. J. Biol. Chem. 2001; 276: 24565-24573Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Anti-GHRcyt-AL37 was raised against a bacterially expressed glutathione S-transferase fusion with human GHR residues 271–620, as described (47Jiang J. Liang L. Kim S.O. Zhang Y. Mandler R. Frank S.J. Biochem. Biophys. Res. Commun. 1998; 253: 774-779Crossref PubMed Scopus (50) Google Scholar). Anti-GHRcyt was raised against a bacterially expressed maltose-binding protein fusion with human GHR residues 317–620, as described (48Frank S.J. Gilliland G. Van Epps C. Endocrinology. 1994; 135: 148-156Crossref PubMed Scopus (48) Google Scholar). In some experiments, the antisera were affinity purified for immunoblotting (as in Ref. 48Frank S.J. Gilliland G. Van Epps C. Endocrinology. 1994; 135: 148-156Crossref PubMed Scopus (48) Google Scholar). In others, they were not purified and gave rise to some nonspecific bands, as indicated. For immunofluorescence microscopy, anti-GHRcyt-AL47 was purified as follows. An IgG fraction was purified using protein A-Sepharose (nProtein A-Sepharose 4 Fast Flow; Amersham Biosciences). Bound antibodies were acideluted with 0.1 m glycine-HCl, pH 2.8, and neutralized with one-tenth volume Tris-HCl (1 m), pH 9.0. NaCl was then added to 150 mm. The eluted antibodies were then adsorbed against Escherichia coli acetone powder, as described (49Harlow E. Lane M.D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 632-633Google Scholar). Cell Stimulation, Protein Extraction, Immunoprecipitation, Electrophoresis, and Immunoblotting—Serum starvation of all cell lines was accomplished by substitution of 0.5% (w/v) bovine serum albumin (fraction V; Roche Applied Science) for serum in their respective culture media for 16–20 h prior to experiments. Stimulations were performed at 37 °C. Pretreatment of each cell line was as follows: 1) for 3T3-F442A, PS1/2 knock-out, and PS1/2 WT cells, vehicle control and/or inhibitors were added 30 min prior to stimulation; 2) for HEK-293, vehicle control and/or inhibitors were added 4 h prior to PMA stimulation; 3) for γ2A-rbGHR, vehicle control and/or inhibitors were added 30–60 min prior to PMA stimulation; and 4) for R+T mouse fibroblasts, vehicle control and/or inhibitors were added 90 min prior to PMA stimulation. Details of the PMA (at 1 μg/ml) treatment protocol have been described (12Goldsmith J.F. Lee S.J. Jiang J. Frank S.J. Am. J. Physiol. 1997; 273: E932-E941PubMed Google Scholar, 13Alele J. Jiang J. Goldsmith J.F. Yang X. Maheshwari H.G. Black R.A. Baumann G. Frank S.J. Endocrinology. 1998; 139: 1927-1935Crossref PubMed Google Scholar, 14Guan R. Zhang Y. Jiang J. Baumann C.A. Black R.A. Baumann G. Frank S.J. Endocrinology. 2001; 142: 1137-1147Crossref PubMed Scopus (41) Google Scholar, 15Zhang Y. Guan R. Jiang J. Kopchick J.J.

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