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• W2076439922 abstract "Substance P analogues including [d-Arg1,d-Phe5,d-Trp7,9,Leu11]substance P (SpD) act as “broad spectrum neuropeptide antagonists” and are potential anticancer agents that inhibit the growth of small cell lung cancer cells in vitro and in vivo. However, their mechanism of action is controversial and not fully understood. Although these compounds block bombesin-induced mitogenesis and signal transduction, they also have agonist activity. The mechanism underlying this agonist activity was examined. SpD binds to the ligand-binding site of the bombesin/gastrin-releasing peptide receptor and blocks the bombesin-stimulated increase in [Ca2+]i within the same concentration range that causes sustained activation of c-Jun N-terminal kinase and extracellular signal-regulated protein kinase (ERK). The activation of c-Jun N-terminal kinase by SpD and bombesin is blocked by dominant negative inhibition of Gα12. The ERK activation by SpD is pertussis toxin-sensitive in contrast to ERK activation by bombesin, which is pertussis toxin-insensitive but dependent on epidermal growth factor receptor phosphorylation. SpD does not simply act as a partial agonist but differentially modulates the activation of the G-proteins Gα12, Gi, and Gq compared with bombesin. This unique ability allows the bombesin receptor to couple to Gi and at the same time block receptor activation of Gq. Our results provide direct evidence that SpD is acting as a “biased agonist” and that this has physiological relevance in small cell lung cancer cells. This validation of the concept of biased agonism has important implications in the development of novel pharmacological agents to dissect receptor-mediated signal transduction and of highly selective drugs to treat human disease. Substance P analogues including [d-Arg1,d-Phe5,d-Trp7,9,Leu11]substance P (SpD) act as “broad spectrum neuropeptide antagonists” and are potential anticancer agents that inhibit the growth of small cell lung cancer cells in vitro and in vivo. However, their mechanism of action is controversial and not fully understood. Although these compounds block bombesin-induced mitogenesis and signal transduction, they also have agonist activity. The mechanism underlying this agonist activity was examined. SpD binds to the ligand-binding site of the bombesin/gastrin-releasing peptide receptor and blocks the bombesin-stimulated increase in [Ca2+]i within the same concentration range that causes sustained activation of c-Jun N-terminal kinase and extracellular signal-regulated protein kinase (ERK). The activation of c-Jun N-terminal kinase by SpD and bombesin is blocked by dominant negative inhibition of Gα12. The ERK activation by SpD is pertussis toxin-sensitive in contrast to ERK activation by bombesin, which is pertussis toxin-insensitive but dependent on epidermal growth factor receptor phosphorylation. SpD does not simply act as a partial agonist but differentially modulates the activation of the G-proteins Gα12, Gi, and Gq compared with bombesin. This unique ability allows the bombesin receptor to couple to Gi and at the same time block receptor activation of Gq. Our results provide direct evidence that SpD is acting as a “biased agonist” and that this has physiological relevance in small cell lung cancer cells. This validation of the concept of biased agonism has important implications in the development of novel pharmacological agents to dissect receptor-mediated signal transduction and of highly selective drugs to treat human disease. small cell lung cancer c-Jun N-terminal kinase extracellular signal-regulated protein kinase [d-Arg1,d-Phe5,d-Trp7,9,Leu11]substance P gastrin-releasing peptide Dulbecco's modified Eagle's medium hemagglutinin fetal calf serum polyacrylamide gel electrophoresis epidermal growth factor Neuropeptides have been implicated in the pathogenesis of a number of human disease states including inflammatory disease, cardiovascular disease, and cancer (1Rozengurt E. Am. J. Physiol. 1998; 275: G177-G182PubMed Google Scholar). Neuropeptides including bombesin are autocrine growth factors for a number of cancers including breast, prostate, and small cell lung cancer (SCLC)1 (2Moody T.W. Cuttitta F. Life Sci. 1993; 52: 1161-1173Crossref PubMed Scopus (92) Google Scholar, 3Wang Q.J. Knezetic J.A. Schally A.V. Pour P.M. Adrian T.E. Int. J. Cancer. 1996; 68: 528-534Crossref PubMed Google Scholar, 4Moody T.W. Pert C.B. Gazdar A.F. Carney D.N. Minna J.D. Science. 1981; 214: 1246-1248Crossref PubMed Scopus (392) Google Scholar, 5Cuttitta F. Carney J. Mulshine J. Moody T.W. Fedorko J. Fischler A. Minna J.D. Nature. 1985; 316: 823-826Crossref PubMed Scopus (1149) Google Scholar). In particular, neuropeptides and their receptors are the principle driving force behind one of the most clinically aggressive cancers, SCLC. SCLC cells sustain their growth in part as a result of multiple autocrine and paracrine loops involving calcium-mobilizing neuropeptides (6Sethi T. Rozengurt E. Cancer Res. 1991; 51: 3621-3623PubMed Google Scholar, 7Sethi T. Langdon S.P. Smyth J.S. Rozengurt E. Cancer Res. 1992; 52 (suppl.): 2737-2742Google Scholar). SCLC provides a paradigm for the investigation of neuropeptide-mediated growth. Modulating neuropeptide-induced signal transduction may therefore have important implications in the treatment of a number of human diseases. Neuropeptides are a structurally diverse group of hormones and neurotransmitters that bind to a related subfamily of G-protein-coupled receptors. Predominately, these receptors couple to Gq to elicit phospholipase C-β activation and subsequent production of diacylglycerol and phosphatidylinositol 1,4,5-trisphosphate leading to protein kinase C activation and Ca2+ release (8Offermans S. Heiler E. Spicher K. Schultz G. FEBS Lett. 1994; 349: 201-204Crossref PubMed Scopus (64) Google Scholar, 9Jian X. Sainz E. Clark W.A. Jensen R.T. Battey J.K. Northup J.K. J. Biol. Chem. 1999; 274: 11573-11581Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 10Charlesworth A. Broad S. Rozengurt E. Oncogene. 1996; 12: 1337-1345PubMed Google Scholar). These receptors also couple to G12 to elicit c-Jun N-terminal kinase (JNK) activation and Rho-dependent activation of stress fiber formation via tyrosine phosphorylation of a number of tyrosine kinases including FAK, paxillin, and p130cas(11Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3824) Google Scholar, 12Buhl A.M. Johnson N.L. Dhanasekaran N. Johnson G.L. J. Biol. Chem. 1995; 270: 24631-24634Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 13Needham L.K. Rozengurt E. J. Biol. Chem. 1998; 273: 14626-14632Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 14Gohla A. Offermanns S. Wilkie T.M. Schultz G. J. Biol. Chem. 1999; 274: 17901-17907Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Bombesin and other neuropeptides have also been shown to activate the ERK pathway leading to the stimulation of immediate early genes and proliferation (10Charlesworth A. Broad S. Rozengurt E. Oncogene. 1996; 12: 1337-1345PubMed Google Scholar, 15Rozengurt E. Sinnet-Smith J. Prog. Nucleic Acid Res. Mol. Biol. 1988; 35: 261-295Crossref PubMed Scopus (57) Google Scholar, 16Slice L. Walsh J.H. Rozengurt E. J. Biol. Chem. 1999; 274: 27562-27566Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Analogues of substance P, [d-Arg1,d-Phe5,d-Trp7,9,Leu11]substance P (SpD) and [Arg6,d-Trp7,9,NmePhe8]substance P (6Sethi T. Rozengurt E. Cancer Res. 1991; 51: 3621-3623PubMed Google Scholar, 7Sethi T. Langdon S.P. Smyth J.S. Rozengurt E. Cancer Res. 1992; 52 (suppl.): 2737-2742Google Scholar, 8Offermans S. Heiler E. Spicher K. Schultz G. FEBS Lett. 1994; 349: 201-204Crossref PubMed Scopus (64) Google Scholar, 9Jian X. Sainz E. Clark W.A. Jensen R.T. Battey J.K. Northup J.K. J. Biol. Chem. 1999; 274: 11573-11581Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 10Charlesworth A. Broad S. Rozengurt E. Oncogene. 1996; 12: 1337-1345PubMed Google Scholar, 11Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3824) Google Scholar) can inhibit neuropeptide-stimulated Ca2+mobilization, tyrosine phosphorylation, and ERK activation (17Seckl M.J. Newman R.H. Freemont P.S. Rozengurt E. J. Cell. Physiol. 1995; 163: 87-95Crossref PubMed Scopus (18) Google Scholar, 18Seckl M.J. Higgins T. Rozengurt E. J. Biol. Chem. 1996; 271: 29453-29460Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 19Seckl M.J. Higgins T. Widmer F. Rozengurt E. Cancer Res. 1997; 57: 51-54PubMed Google Scholar). Crucially, SpD and [Arg6,d-Trp7,9,NmePhe8]substance P inhibit SCLC cell growth in vivo and in vitro(7Sethi T. Langdon S.P. Smyth J.S. Rozengurt E. Cancer Res. 1992; 52 (suppl.): 2737-2742Google Scholar, 20Woll P.J. Rozengurt E. Proc. Nat. Acad. Sci. U. S. A. 1988; 85: 1859-1863Crossref PubMed Scopus (169) Google Scholar) and stimulate SCLC cell apoptosis (21MacKinnon A.C. Armstrong R.A. Waters C. Cummings J. Smyth J.F. Haslett C. Sethi T. Br. J. Cancer. 1999; 80: 1026-1034Crossref PubMed Scopus (31) Google Scholar, 22Reeve J.G. Bleehen N.M. Biochem. Biophys. Res. Commun. 1994; 199: 1313-1319Crossref PubMed Scopus (57) Google Scholar). Substance P analogues are about to enter phase II clinical investigation for the treatment of SCLC and could provide a novel form of therapy for other neuroendocrine tumors in addition to SCLC (23MacKinnon A.C. Waters C. Rahman I. Harani N. Rintoul R. Haslett C. Sethi T. Br. J. Cancer. 2000; 83: 941-948Crossref PubMed Scopus (12) Google Scholar). Hence understanding the mechanism of action of this class of compound is attracting considerable interest and is critical for future drug development. Substance P analogues were characterized originally as “broad spectrum neuropeptide antagonists” (7Sethi T. Langdon S.P. Smyth J.S. Rozengurt E. Cancer Res. 1992; 52 (suppl.): 2737-2742Google Scholar, 17Seckl M.J. Newman R.H. Freemont P.S. Rozengurt E. J. Cell. Physiol. 1995; 163: 87-95Crossref PubMed Scopus (18) Google Scholar, 18Seckl M.J. Higgins T. Rozengurt E. J. Biol. Chem. 1996; 271: 29453-29460Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 19Seckl M.J. Higgins T. Widmer F. Rozengurt E. Cancer Res. 1997; 57: 51-54PubMed Google Scholar). However, the precise mechanism of action of these compounds seems more complex and remains unclear and contentious (23MacKinnon A.C. Waters C. Rahman I. Harani N. Rintoul R. Haslett C. Sethi T. Br. J. Cancer. 2000; 83: 941-948Crossref PubMed Scopus (12) Google Scholar). Studies in Swiss 3T3 cells suggested that substance P analogues competitively inhibit the binding of neuropeptides to their receptors, accounting for the inhibition of neuropeptide-stimulated signal transduction (17Seckl M.J. Newman R.H. Freemont P.S. Rozengurt E. J. Cell. Physiol. 1995; 163: 87-95Crossref PubMed Scopus (18) Google Scholar, 18Seckl M.J. Higgins T. Rozengurt E. J. Biol. Chem. 1996; 271: 29453-29460Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Substance P analogues were thought to inhibit SCLC cell growth by competitively inhibiting the mitogenic effects of autocrine neuropeptides (17Seckl M.J. Newman R.H. Freemont P.S. Rozengurt E. J. Cell. Physiol. 1995; 163: 87-95Crossref PubMed Scopus (18) Google Scholar, 18Seckl M.J. Higgins T. Rozengurt E. J. Biol. Chem. 1996; 271: 29453-29460Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 19Seckl M.J. Higgins T. Widmer F. Rozengurt E. Cancer Res. 1997; 57: 51-54PubMed Google Scholar, 20Woll P.J. Rozengurt E. Proc. Nat. Acad. Sci. U. S. A. 1988; 85: 1859-1863Crossref PubMed Scopus (169) Google Scholar). However, the SCLC cell growth inhibitory and proapoptotic activities of substance P analogues are not reversed by supramaximal saturating concentrations of neuropeptides (21MacKinnon A.C. Armstrong R.A. Waters C. Cummings J. Smyth J.F. Haslett C. Sethi T. Br. J. Cancer. 1999; 80: 1026-1034Crossref PubMed Scopus (31) Google Scholar). Furthermore, substance P analogues themselves activate JNK and potentiate bradykinin-induced edema formation in rabbit skin (21MacKinnon A.C. Armstrong R.A. Waters C. Cummings J. Smyth J.F. Haslett C. Sethi T. Br. J. Cancer. 1999; 80: 1026-1034Crossref PubMed Scopus (31) Google Scholar, 23MacKinnon A.C. Waters C. Rahman I. Harani N. Rintoul R. Haslett C. Sethi T. Br. J. Cancer. 2000; 83: 941-948Crossref PubMed Scopus (12) Google Scholar, 24Jarpe M.B. Knall C. Mitchell F.M. Buhl A. Duzic E. Johnson G.L. J. Biol. Chem. 1998; 273: 3097-3104Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Further studies have shown that although SpD irreversibly blocks bombesin-induced phospholipase C-β activation and mitogenesis and reversibly inhibits ERK activation by bombesin (25Mitchell F.M. Heasley L.E. Qian N.X. Zamarripa J. Johnson G.L. J. Biol. Chem. 1995; 270: 8623-8628Crossref PubMed Scopus (43) Google Scholar), SpD augments Raf-1 and ERK activation by high concentrations of bombesin (25Mitchell F.M. Heasley L.E. Qian N.X. Zamarripa J. Johnson G.L. J. Biol. Chem. 1995; 270: 8623-8628Crossref PubMed Scopus (43) Google Scholar). This suggests that the GRP receptor may still be capable of signaling even when bombesin-induced phospholipase C activation is fully blocked. Thus in addition to its well described antagonist activity, SpD also has agonist activity. The ability of a receptor to have more than one active state and interact with multiple G-proteins to produce cellular responses has been described in a variety of receptor systems and has been termed “agonist-receptor trafficking” (26Kenakin T. Trends. Pharmacol. Sci. 1995; 16: 232-238Abstract Full Text PDF PubMed Scopus (580) Google Scholar, 27Berg K.A. Maayani S. Goldfarb J. Scaramellini C. Leff P. Clarke W.P. Mol. Pharmacol. 1998; 54: 94-104Crossref PubMed Scopus (445) Google Scholar). Jarpe et al.(24Jarpe M.B. Knall C. Mitchell F.M. Buhl A. Duzic E. Johnson G.L. J. Biol. Chem. 1998; 273: 3097-3104Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) hypothesized that SpD may act as an agonist at GRP receptors, activating the G12 family of guanine-nucleotide binding proteins while blocking signal transduction via Gqand proposed a novel pharmacological term, “biased agonism,” to describe this. Agents acting by the mechanism of biased agonism that selectively activate G-proteins would have enormous pharmacological and clinical importance and could become valuable tools in the dissection of signal transduction pathways downstream of receptors. However, the hypothesis of biased agonism was challenged by Sinnett-Smith et al. (28Sinnett-Smith J. Santiskulvong C. Duque J. Rozengurt E. J. Biol. Chem. 2000; 275: 30644-30652Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), who concluded that substance P analogues acted primarily as antagonists of neuropeptide receptors, blocking signal transduction via both G12 and Gq, and that any agonist activity was caused by partial agonism. The experiments presented here were designed to examine the mechanism underlying the agonist activity of SpD and to investigate specifically the validity of the biased agonist hypothesis. Rat-1a cells and rat-1a cells stably expressing the mouse bombesin/GRP receptor (BOR-15) were established by the Imperial Cancer Research Fund (London). COS-7 cells and NCI-H345 SCLC cells were purchased from the American Type Tissue Culture Collection (Manassas, VA). Native Balb 3T3 cells and Balb 3T3 cells expressing the mutant bombesin receptors (5ET4 and R288H) were supplied by Dr. J. F. Battey (National Institutes of Health, Rockville, Maryland). Dominant negative G-protein α subunit cDNAs G12(G228A) and G13 (G225A) in the pCIS transfection vector were supplied kindly by Dr. Stefan Offermans (Freie Universitaet Berlin, Germany). RPMI 1640 medium, Dulbecco's modified Eagle's medium (DMEM), bombesin, anti-phospho-ERK1/2 monoclonal antibody, myelin basic protein, and SpD were from Sigma; [γ-33P]ATP (3000 Ci/mmol), [γ-32P]ATP (3000 Ci/mmol), and [125I]-GRP (2000 Ci/mmol) were fromAmersham Pharmacia Biotech; anti-ERK2, anti-JNK1, anti-phospho-JNK1/2, anti-HA, polyclonal antibodies, and glutathione S-transferase-c-Jun (79) were from Insight Biotechnology (Wembley, UK); pertussis toxin was from Alexis (Nottingham, UK); and AG1478 and fura-2-tetraacetoxymethyl ester (Fura-2-AME) were from Calbiochem (Nottingham UK). RC-3095 was a kind gift from Dr. A.V. Schally (Section of Experimental Medicine, Tulane University School of Medicine). All other reagents were of the purest grade available. Rat-1a, BOR-15, and COS-7 cells were maintained in DMEM containing 10% (v/v) fetal calf serum, 50 units/ml penicillin, 50 µg/ml streptomycin, and 5 µg/mll-glutamine. G418 (400 µg/ml) was added to BOR-15 cells. NCI-H345 SCLC cells were cultured in RPMI 1640 medium with 25 mm HEPES supplemented with 10% (v/v) fetal calf serum, 50 units/ml penicillin, 50 µg/ml streptomycin and 5 µg/mll-glutamine in a humidified atmosphere of 5% CO2/95% air at 37 °C. For experimental purposes, the cells were grown in SITA medium consisting of RPMI 1640 medium supplemented with 30 nm selenium, 5 µg/ml insulin, 10 µg/ml transferrin, and 0.25% (w/v) bovine serum albumin. SCLC cells were made quiescent in serum-free RPMI 1640 medium containing 0.25% (w/v) bovine serum albumin. COS-7 cells transiently expressing the dominant negative Gα12 (G228A) and Gα13(G225A) and HA-JNK1 were generated using LipofectAMINE (Life Technologies, Inc.). The cells were transfected with 5 µg of pCIS-lacZ, G228A, or G225A and HA-JNK1 and grown for 1 day in DMEM containing 10% FCS. The cells were then split 1:4 and grown for 1 day in 10% (v/v) FCS until 70% confluent in 90-mm tissue culture plates. The cells were then made quiescent by incubation for 1 day in 0.1% (v/v) FCS prior to JNK analysis. [Ca2+]i was measured using the fluorescent Ca2+ probe Fura-2-AME as described previously (27Berg K.A. Maayani S. Goldfarb J. Scaramellini C. Leff P. Clarke W.P. Mol. Pharmacol. 1998; 54: 94-104Crossref PubMed Scopus (445) Google Scholar). Briefly, cells in 90-mm dishes were made quiescent overnight in DMEM containing 0.1% (v/v) FCS, washed once, and trypsinized. The cells were washed and suspended in calcium-free Hanks' modified electrolyte solution and incubated with 2 µm Fura-2-AME for 10 min at 37 °C. The cells were suspended in Hanks' modified electrolyte solution containing calcium and transferred to a cuvette maintained at 37 °C. Agonists were added as described in the figure legends. Ratiometric fluorescence was monitored in a PerkinElmer fluorometric spectrophotometer with dual excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. The [Ca2+]i was calculated according to the equation [Ca2+]i = K(F −F min)/(F max −F), where F is the ratio of the unknown sample, F max is the ratio after the addition of 0.2% Triton X-100, and F min is the ratio after Ca2+ chelation with 10 mm EGTA. K is the dissociation constant for Fura-2, which is 224 nm (29Tsien R.Y. Pozzan T. Rink T.J. Nature. 1992; 295: 68-71Crossref Scopus (774) Google Scholar). Quiescent cell cultures were treated as described in the figure legends and lysed at 4 °C in 0.5 ml of lysis buffer containing 25 mm HEPES, pH 7.4, 0.3m NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5% Triton X-100, 20 mmβ-glycerophosphate, 0.5 mm dithiothreitol, 1 mm sodium orthovanadate, and protease inhibitors (the protease inhibitor mixture was from Roche Molecular Biochemicals and prepared as per manufacturer instructions). The lysates were clarified by centrifugation, equilibrated for protein, and ERK2-immunoprecipitated using 3 µg of anti-ERK2 polyclonal antibody coupled to Sepharose (Santa Cruz Biotechnology). An aliquot of lysate was retained and boiled in Laemmli SDS-PAGE loading buffer for future Western analysis. Immune complexes from cell lysates containing 400 µg of protein were washed three times at 4 °C in 20 mm HEPES, pH 7.4, containing 50 mm NaCl, 2.5 mm MgCl2, and 0.1 mm EDTA by repeated centrifugation and once at 4 °C in kinase buffer (20 mm HEPES, pH 7.4, containing 0.5 mm NaF, 7.5 mmMgCl2, 0.2 mm EGTA, 2 mmdithiothreitol, 10 mm β-glycerophosphate, and 0.5 mm sodium orthovanadate). Kinase activity was estimated in 25 µl of kinase buffer containing 100 µm ATP, 1 µCi of [γ-33P]ATP (3000 Ci/mmol), and 10 µg of myelin basic protein. The reaction was carried out for 20 min at 30 °C and terminated by spotting the supernatant onto P81 phosphocellulose paper. The papers were washed three times in 0.5% (v/v) phosphoric acid and dried briefly in acetone. The results are expressed as specific disintegrations/min bound or -fold increase over basal. Cell lysates were generated as described for ERK2. After immunoprecipitation of JNK1 from whole-cell lysates, kinase activity was carried out as described (21MacKinnon A.C. Armstrong R.A. Waters C. Cummings J. Smyth J.F. Haslett C. Sethi T. Br. J. Cancer. 1999; 80: 1026-1034Crossref PubMed Scopus (31) Google Scholar) using 20 µm ATP, 1 µCi of [32P]ATP (3000 Ci/mmol), and 1 µg of glutathioneS-transferase-c-Jun (79) substrate. Phosphorylated c-Jun was identified from autoradiographs of Coomassie Blue-stained SDS-PAGE gels and quantified by phosphorimaging. HA-JNK1 was precipitated from transiently transfected COS-7 cells using 2 µg of anti-HA antibody coupled to Sepharose (Santa Cruz Biotechnology). JNK activity was determined as described above. Whole-cell lysates were normalized for protein concentration (typically 1.0–2.0 mg/ml) and denatured by boiling (5 min) in SDS-PAGE loading buffer. 20 µl of lysate/lane was resolved on 12% SDS-PAGE gels and electroblotted onto nitrocellulose membranes. The membranes were blocked in 5% nonfat milk in PBS containing 0.05% Tween 20. ERK1/2 and JNK1/2phosphorylation was determined using 1:1000 dilution of the primary antibody followed by the appropriate horseradish peroxidase-labeled goat IgG (DAKO) diluted 1:5000. Bands were visualized using enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech). [125I]-GRP receptor binding was carried out in confluent and quiescent cultures of BOR-15 cells exactly as described (10Charlesworth A. Broad S. Rozengurt E. Oncogene. 1996; 12: 1337-1345PubMed Google Scholar). The binding parameters K d andB max were calculated according to the method described by Scatchard et al. (30Scatchard G. Ann. N. Y. Acad. Sci. 1949; 51: 660-666Crossref Scopus (17798) Google Scholar). The IC50(concentration of drug displacing 50% specific binding) was converted to the inhibitory constant (K i), whereK i = IC50/(1 + [ligand]/K d) (31Cheng Y.C. Prusoff W.H. Biochem. Pharmacol. 1973; 22: 3099-3108Crossref PubMed Scopus (12265) Google Scholar). The mechanism underlying the agonist activity of SpD is unclear and controversial. SpD can bind to multiple characterized and uncharacterized G-protein-linked receptors. To define precisely the mechanism underlying the agonist activity of SpD, we used a rat-1a fibroblast model system (into which the bombesin receptor is transfected) to mediate SpD activity. Bombesin and other Ca2+-mobilizing neuropeptides (vasopressin, neurotensin, bradykinin, and gastrin), fail to mobilize intracellular Ca2+ in native rat-1a fibroblasts, suggesting the absence of these receptors in this cell line (Fig.1A). In rat-1a cells stably expressing the bombesin/GRP receptor (BOR-15), bombesin induces a marked and rapid increase in [Ca2+]i, which was blocked by SpD (Fig. 1 B). In addition, SpD at concentrations as high as 100 µm failed to mobilize intracellular calcium in rat-1a or BOR-15 cells (Fig. 1 C). In BOR-15 cells, bombesin induced a concentration-dependent increase in [Ca2+]i with EC50 = 1.3 ± 0.2 nm. These results are similar to those seen for bombesin stimulation of [Ca2+]i in Swiss 3T3 cells (7Sethi T. Langdon S.P. Smyth J.S. Rozengurt E. Cancer Res. 1992; 52 (suppl.): 2737-2742Google Scholar, 17Seckl M.J. Newman R.H. Freemont P.S. Rozengurt E. J. Cell. Physiol. 1995; 163: 87-95Crossref PubMed Scopus (18) Google Scholar). SpD (30 µm) shifted the bombesin concentration response curve to the right (Fig. 1 D,inset) with a K i for the inhibition of bombesin-stimulated Ca2+ flux of 6.4 ± 0.7 µm. These results are similar to those seen for SpD inhibition of both bombesin-induced stimulation of [Ca2+]i and DNA synthesis in Swiss 3T3 cells 2A. C. MacKinnon and T. Sethi, unpublished observations. (7Sethi T. Langdon S.P. Smyth J.S. Rozengurt E. Cancer Res. 1992; 52 (suppl.): 2737-2742Google Scholar, 17Seckl M.J. Newman R.H. Freemont P.S. Rozengurt E. J. Cell. Physiol. 1995; 163: 87-95Crossref PubMed Scopus (18) Google Scholar) and for the inhibition of [125I]-GRP receptor binding (Fig. 5).Figure 5[125I]-GRP receptor binding to BOR-15 cells. A, confluent cultures of BOR-15 cells (30-mm dishes) were incubated with SpD (1 nm-100 µm) and [125I]-GRP (0.01 µCi and 1 nm unlabeled GRP) in DMEM containing 1 mg/ml bovine serum albumin for 30 min at 37 °C. Cells were washed three times in cold PBS, solubilized in 0.1 n NaOH/2% NaCO3/1% SDS, and counted for [125I] in a beta scintillation counter. Nonspecific binding was defined in the presence of 1 µm bombesin and was routinely 35–45% of the total. Inset, Scatchard analysis of [125I]-GRP binding in BOR-15 cells. BOR-15 cells were incubated with [125I]-GRP (0.01 µCi) and unlabeled GRP (0.01–30 nm) for 30 min at 37 °C. Bound radioactivity was determined as described for A. Results are presented as a Scatchard plot (28Sinnett-Smith J. Santiskulvong C. Duque J. Rozengurt E. J. Biol. Chem. 2000; 275: 30644-30652Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) representative of four independent experiments. BOR-15 cells bound [125I]-GRP with an affinity (K d) of 0.48 ± 0.11 nm and aB max of 0.72 ± 0.08 × 105 binding sites/cell. B, desensitization of the GRP receptor after bombesin or SpD pretreatment. Confluent cultures of BOR-15 cells were washed once in PBS and incubated in DMEM containing 1 mg/ml bovine serum albumin and either 30 nmbombesin (●) or 30 µm SpD (■) for various times as indicated. Cells were then washed three times in PBS and incubated with [125I]-GRP (0.01 µCi and 1 nm unlabeled GRP) as described for A. Results are expressed as specific binding (disintegrations/min) and represent the mean ± S.E. of three experiments performed in triplicate.View Large Image Figure ViewerDownload (PPT) The addition of SpD for 10 min causes a marked concentration-dependent increase in ERK activity in quiescent rat-1a fibroblasts expressing the bombesin receptor (BOR-15 cells), in contrast to the untransfected rat-1a cells (Fig. 2). In BOR-15 cells, bombesin stimulation of ERK activity was seen in the nm range, maximal at 30 nm (EC50 = 5.9 ± 1.8 nm, n = 4). SpD-induced stimulation of ERK activity in BOR-15 cells was evident at 3 µm, maximal at 10 µm (EC50 = 4.19 ± 0.6 µm, n = 3) (Fig. 2). To ensure that this effect was not a result of clonal selection, cells were transiently transfected with the mouse bombesin receptor, and similar results were obtained (results not shown). The stimulation of ERK occurred within the same concentration range at which SpD inhibits bombesin-stimulated Ca2+ release. The time course of ERK activation by SpD is different from that of bombesin (Fig. 3). ERK1/2phosphorylation as an indication of ERK activity was assessed by immunoblotting with a phosphorylation state-specific monoclonal antibody to ERK1/2 and showed quantitatively similar results to that of the immunoprecipitation kinase assay (data not shown). SpD-induced ERK1/2 phosphorylation was evident at 5 min, reached a maximum at 10 min, and persisted for over 60 min. The time course of ERK1/2 activation by SpD was slower in onset and more sustained in comparison with that of bombesin, which returned to control levels by 30 min (Fig. 3). This suggests that SpD and bombesin differentially regulate signal transduction pathways leading to ERK activation. SpD- and bombesin-induced ERK activity in BOR-15 cells was inhibited by two specific GRP/bombesin receptor antagonists, [Leu13(CH2NH)-Leu14]bombesin and RC-3095 (32Koppan M. Halmos G. Arencibia J.M. Lamharzi N. Schally A.V. Cancer. 1998; 83: 1335-1343Crossref PubMed Scopus (47) Google Scholar). Pretreatment of BOR-15 cells for 30 min with [Leu13(CH2NH)-Leu14]bombesin (30 nm) or 1 µm RC-3095 (1 µm) completely blocked the ERK activity stimulated by either bombesin (30 nm) or SpD (30 µm, Fig.4A). These results suggest that SpD-induced ERK activation is GRP receptor-dependent and occurs in the absence of a calcium signal. Bombesin did not stimulate an increase in [Ca2+]i in Balb 3T3 cells. In Balb 3T3 cells expressing the wild-type GRP receptor (5ET4), 3 nm bombesin stimulated a maximal increase in [Ca2+]i of 375 nm. However, in Balb 3T3 cells expressing the mutated receptor (R288H), 30 nm bombesin did not increase [Ca2+]i with 100 nm bombesin increasing [Ca2+]i by 47 nm(results not shown). This is consistent with previously reported data showing that Arg-288 in the bombesin/GRP receptor is critical for high affinity bombesin binding and that the R288H mutation does not affect GRP receptor expression or conformation and otherwise the R288H mutant GRP-receptor is fully functional (33Akeson M. Sainz E. Mantey S.A Jensen R.T. Battey J.F. J. Biol. Chem. 1997; 272: 17405-17409Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 34Donohue P.J. Sainz E. Akeson M. Kroog G.S. Mantey S.A. Battey J.F. Jensen R.T. Northup J.K. Biochemistry. 1999; 38: 9366-9372Crossref PubMed Scopus (17) Google Scholar). 5ET4 or R288H cells were stimulated with bombesin (3 nm) or SpD (0.3–30 µm) for 10 min. Bombesin (3 nm) caused a 2.6-fo" @default.
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• W2076439922 title "Bombesin and Substance P Analogues Differentially Regulate G-protein Coupling to the Bombesin Receptor" @default.
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