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• W2056528957 abstract "We have previously mapped the agonist-induced phosphorylation of the rat lutropin/choriogonadotropin receptor (rLHR) to a locus of four serines (Ser635, Ser639, Ser649, and Ser652) located in the C-terminal tail. The removal or mutation of this locus delays the time course of agonist-induced uncoupling of the rLHR from its effector system without affecting the overall magnitude of uncoupling, and it retards the endocytosis of the agonist-receptor complex.We have now prepared and analyzed four new rLHR mutants in which each of these serines were individually mutated to alanines. The data presented show that each mutation reduces agonist-promoted rLHR phosphorylation by 20–40%. Mutation of Ser635 or Ser639 delayed the time course of agonist-induced uncoupling to about the same extent as the simultaneous mutation of all four serines. Mutation of Ser635 or Ser639 also retarded agonist-induced internalization, but the magnitude of this decrease was less than that induced by the simultaneous mutation of all four serines. Mutation of Ser649 had no effect on agonist-induced uncoupling but retarded agonist-induced internalization to the same extent as the simultaneous mutation of all four serines. Mutation of Ser652 has little or no effect on either of these two parameters.Co-transfection studies with dominant-negative arrestins and dominant-negative dynamin reveal that, despite differences in their rates of internalization, rLHR-wild-type, rLHR-S639A, and rLHR-S649A are internalized by an arrestin- and dynamin-dependent pathway.These data show that the structural requirements needed for the agonist-induced uncoupling and internalization of the rLHR are distinct. We have previously mapped the agonist-induced phosphorylation of the rat lutropin/choriogonadotropin receptor (rLHR) to a locus of four serines (Ser635, Ser639, Ser649, and Ser652) located in the C-terminal tail. The removal or mutation of this locus delays the time course of agonist-induced uncoupling of the rLHR from its effector system without affecting the overall magnitude of uncoupling, and it retards the endocytosis of the agonist-receptor complex. We have now prepared and analyzed four new rLHR mutants in which each of these serines were individually mutated to alanines. The data presented show that each mutation reduces agonist-promoted rLHR phosphorylation by 20–40%. Mutation of Ser635 or Ser639 delayed the time course of agonist-induced uncoupling to about the same extent as the simultaneous mutation of all four serines. Mutation of Ser635 or Ser639 also retarded agonist-induced internalization, but the magnitude of this decrease was less than that induced by the simultaneous mutation of all four serines. Mutation of Ser649 had no effect on agonist-induced uncoupling but retarded agonist-induced internalization to the same extent as the simultaneous mutation of all four serines. Mutation of Ser652 has little or no effect on either of these two parameters. Co-transfection studies with dominant-negative arrestins and dominant-negative dynamin reveal that, despite differences in their rates of internalization, rLHR-wild-type, rLHR-S639A, and rLHR-S649A are internalized by an arrestin- and dynamin-dependent pathway. These data show that the structural requirements needed for the agonist-induced uncoupling and internalization of the rLHR are distinct. Phosphorylation of G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G protein-coupled receptor; LHR, lutropin/choriogonadotropin receptor; rLHR, rat LHR; wt, wild-type; GRK, G protein-coupled receptor kinase; LH, lutropin; CG, choriogonadotropin; hCG, human CG; β2AR, β2-adrenergic receptor; oLH, ovine lutropin; HA, hemagglutinin. 1The abbreviations used are: GPCR, G protein-coupled receptor; LHR, lutropin/choriogonadotropin receptor; rLHR, rat LHR; wt, wild-type; GRK, G protein-coupled receptor kinase; LH, lutropin; CG, choriogonadotropin; hCG, human CG; β2AR, β2-adrenergic receptor; oLH, ovine lutropin; HA, hemagglutinin. on serine and/or threonine residues is an important event in agonist-induced desensitization. GPCR phosphorylation by second messenger-dependent kinases attenuates signaling by uncoupling the receptors from their cognate G proteins, whereas phosphorylation by the G protein-coupled receptor kinases (GRKs) facilitates the interaction of the receptors with a family of inhibitory proteins called arrestins (1Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (472) Google Scholar, 2Hausdorff W.P. Caron M.G. Lefkowitz R.J. FASEB J. 1990; 4: 2881-2889Crossref PubMed Scopus (1087) Google Scholar). This phosphorylated receptor-arrestin interaction uncouples the receptors from their cognate G proteins and targets the activated receptor to clathrin-coated pits for subsequent internalization (1Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (472) Google Scholar, 2Hausdorff W.P. Caron M.G. Lefkowitz R.J. FASEB J. 1990; 4: 2881-2889Crossref PubMed Scopus (1087) Google Scholar, 3Goodman J.O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1167) Google Scholar, 4Krupnick J.G. Santini F. Gagnon A.W. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 32507-32512Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Thus, the complex formed by the phosphorylated GPCR and arrestin serves as a common intermediate for the uncoupling of the receptor from its cognate G protein and for receptor internalization. Using human kidney 293 cells stably transfected with the rat lutropin/choriogonadotropin receptor (rLHR) cDNA, we showed that, like many other GPCRs, the rLHR becomes phosphorylated on serine residues when the cells are stimulated with an agonist (lutropin (LH) or choriogonadotropin (CG)) (5Hipkin R.W. Sánchez-Yagüe J. Ascoli M. Mol. Endocrinol. 1993; 7: 823-832Crossref PubMed Scopus (44) Google Scholar). The identity of the kinases that mediate the agonist-induced phosphorylation of the rLHR is not known; however, neither kinase A nor kinase C can fully account for the agonist-induced phosphorylation of the rLHR (5Hipkin R.W. Sánchez-Yagüe J. Ascoli M. Mol. Endocrinol. 1993; 7: 823-832Crossref PubMed Scopus (44) Google Scholar, 6Hipkin R.W. Wang Z. Ascoli M. Mol. Endocrinol. 1995; 9: 151-158Crossref PubMed Google Scholar). The involvement of one of the GRKs in the agonist-induced phosphorylation of the rLHR is suggested by the finding that overexpression of GRK2, GRK4, or GRK6 enhances agonist-induced phosphorylation. 2M. d. F. M. Lazari and M. Ascoli, unpublished observations. 2M. d. F. M. Lazari and M. Ascoli, unpublished observations. A GRK-catalyzed phosphorylation of the rLHR is also suggested by functional studies showing that co-transfection of the rLHR with GRK2 or GRK4 diminishes the hCG-induced cAMP response (7Premont R.T. Macrae A.D. Stoffel R.H. Chung N. Pitcher J.A. Ambrose C. Inglese J. MacDonald M.E. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 6403-6410Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). By analogy with what is known about other GPCRs (see above) we proposed that the agonist-induced phosphorylation of the rLHR was responsible for the agonist-induced uncoupling of this receptor from its effector system (5Hipkin R.W. Sánchez-Yagüe J. Ascoli M. Mol. Endocrinol. 1993; 7: 823-832Crossref PubMed Scopus (44) Google Scholar). Further analysis of rLHR mutants truncated at residues 653, 631, or 628 (designated rLHR-t653, rLHR-t631, or rLHR-t628) and a full-length rLHR mutant with multiple serine substitutions (designated rLHR-5S/T→A) mapped the agonist-induced phosphorylation to a cluster of four serine residues (Ser635, Ser639, Ser649, and Ser652) present in the C-terminal tail and established some functional consequences of phosphorylation of this cluster (6Hipkin R.W. Wang Z. Ascoli M. Mol. Endocrinol. 1995; 9: 151-158Crossref PubMed Google Scholar, 8Wang Z. Hipkin R.W. Ascoli M. Mol. Endocrinol. 1996; 10: 748-759PubMed Google Scholar, 9Wang Z. Liu X. Ascoli M. Mol. Endocrinol. 1997; 11: 183-192Crossref PubMed Scopus (50) Google Scholar). Rat LHR-t653, a truncated form of rLHR that retains Ser635, Ser639, Ser649, and Ser652, displays little or no reduction in the agonist-induced phosphorylation, as well as a normal time course and magnitude of agonist-induced uncoupling. On the other hand, rLHR-t631 and rLHR-t628, two truncated forms of the rLHR that lack Ser635, Ser639, Ser649, and Ser652, and a full-length receptor mutant in which these four residues were simultaneously mutated to alanines (i.e.rLHR-5S/T→A) display a 90–100% decrease in agonist-induced phosphorylation and a delay in the rate of agonist-induced uncoupling. The magnitude of agonist-induced uncoupling observed under prolonged agonist stimulation is unaffected, however (8Wang Z. Hipkin R.W. Ascoli M. Mol. Endocrinol. 1996; 10: 748-759PubMed Google Scholar, 9Wang Z. Liu X. Ascoli M. Mol. Endocrinol. 1997; 11: 183-192Crossref PubMed Scopus (50) Google Scholar). It has been known for many years that one of the consequences of agonist binding to the LHR is the endocytosis of the agonist-receptor complex (10Ascoli M. J. Biol. Chem. 1982; 257: 13306-13311Abstract Full Text PDF PubMed Google Scholar). Although the endocytosis of the agonist-bound LHR has been shown to occur via coated pits (11Ghinea N. Vuhai M.T. Groyer-Picard M.-T. Houllier A. Schoëvaërt D. Milgrom E. J. Cell Biol. 1992; 118: 1347-1358Crossref PubMed Scopus (87) Google Scholar), the rate of endocytosis of the agonist-receptor complex is rather slow (half-life of 60–120 min, depending on the cell type; see Refs. 9Wang Z. Liu X. Ascoli M. Mol. Endocrinol. 1997; 11: 183-192Crossref PubMed Scopus (50) Google Scholar, 10Ascoli M. J. Biol. Chem. 1982; 257: 13306-13311Abstract Full Text PDF PubMed Google Scholar, 12Hoelscher S.R. Sairam M.R. Ascoli M. Endocrinology. 1991; 128: 2837-2843Crossref PubMed Scopus (23) Google Scholar, and 13Dhanwada K.R. Vijapurkar U. Ascoli M. Mol. Endocrinol. 1996; 10: 544-554PubMed Google Scholar), and the majority of the agonist-receptor complex is routed to the lysosomes, where both the agonist and the receptor are degraded (11Ghinea N. Vuhai M.T. Groyer-Picard M.-T. Houllier A. Schoëvaërt D. Milgrom E. J. Cell Biol. 1992; 118: 1347-1358Crossref PubMed Scopus (87) Google Scholar, 14Ascoli M. J. Cell Biol. 1984; 99: 1242-1250Crossref PubMed Scopus (72) Google Scholar). This pathway ultimately leads to an agonist-induced reduction in the density of cell surface receptors by routing the receptor to a degradation rather than a recycling pathway (14Ascoli M. J. Cell Biol. 1984; 99: 1242-1250Crossref PubMed Scopus (72) Google Scholar, 15Wang H. Segaloff D.L. Ascoli M. J. Biol. Chem. 1991; 266: 780-785Abstract Full Text PDF PubMed Google Scholar). In keeping with current views on the internalization of other GPCRs, we have shown that the activation of the rLHR is necessary for efficient endocytosis (12Hoelscher S.R. Sairam M.R. Ascoli M. Endocrinology. 1991; 128: 2837-2843Crossref PubMed Scopus (23) Google Scholar, 13Dhanwada K.R. Vijapurkar U. Ascoli M. Mol. Endocrinol. 1996; 10: 544-554PubMed Google Scholar). Moreover, the importance of the phosphorylation of the four-serine locus mentioned above in the endocytosis of the receptor-bound agonist was documented by the finding that cells expressing rLHR-5S/T→A internalize the bound agonist at a slower rate than cells expressing the wild-type rLHR (9Wang Z. Liu X. Ascoli M. Mol. Endocrinol. 1997; 11: 183-192Crossref PubMed Scopus (50) Google Scholar). The experiments presented here were designed to determine which of the four serine residues present in this locus of the rLHR become phosphorylated and to more carefully define the role of each of these residues in the agonist-induced uncoupling and internalization of the rLHR. To this end, we constructed and analyzed four new rLHR mutants in which Ser635, Ser639, Ser649, or Ser652 was individually mutated to an alanine residue in the context of the full-length rLHR. These mutants were analyzed for phosphorylation, uncoupling, and internalization. The cloning of the rat luteal LH/CG receptor cDNA and the template plasmid containing the full-length coding region plus portions of the 5′- and 3′-untranslated regions of the wild-type rLHR cDNA have been described previously (16McFarland K.C. Sprengel R. Phillips H.S. Kohler M. Rosemblit N. Nikolics K. Segaloff D.L. Seeburg P.H. Science. 1989; 245: 494-499Crossref PubMed Scopus (805) Google Scholar). The individual Ser to Ala mutants were constructed using polymerase chain reaction strategies to alter the nucleotides coding for these residues. The sequence of the entire region of each mutant cDNA generated by polymerase chain reaction was verified by automated DNA sequencing. The mutant and wild-type rLHR cDNAs were subcloned into the eukaryotic expression vector pcDNAI/Neo (Invitrogen) for transfection. A plasmid encoding for an HA-tagged dominant-negative mutant form of dynamin (i.e. dynamin-K44A; see Ref. 17Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1037) Google Scholar) was obtained from Sandra Schmid and subcloned into pcDNA3.1 (Invitrogen) for transfection. The expression vectors (all in pcDNA3.1) encoding for β-arrestin, arrestin-3, β-arrestin-V53D, and β-arrestin (319–418) have also been described (4Krupnick J.G. Santini F. Gagnon A.W. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 32507-32512Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Transient co-transfections of human embryonic kidney (293) cells were done using calcium phosphate as described by Chen and Okayama (18Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4820) Google Scholar). Cells plated in 100-mm dishes were transfected when 70–80% confluent using 10 μg of each plasmid. After an overnight incubation, the cells were washed and incubated in growth medium for 1–2 h at 37 °C. The cells were then trypsinized, distributed into 35-mm wells (5–10 × 105 cells/well), and used 24 h later. Stably transfected cell lines were obtained following G418 selection and cloning as described elsewhere (6Hipkin R.W. Wang Z. Ascoli M. Mol. Endocrinol. 1995; 9: 151-158Crossref PubMed Google Scholar, 13Dhanwada K.R. Vijapurkar U. Ascoli M. Mol. Endocrinol. 1996; 10: 544-554PubMed Google Scholar). The establishment and properties of two clonal cell lines expressing rLHR-wt (designated 293L(wt-12) and 293L(wt-17)) and a clonal cell line expressing a mutant in which Ser635, Thr638, Ser639, Ser649, and Ser652 were simultaneously mutated to alanines (designated 293L(5S/T→A-2) have been described (8Wang Z. Hipkin R.W. Ascoli M. Mol. Endocrinol. 1996; 10: 748-759PubMed Google Scholar, 9Wang Z. Liu X. Ascoli M. Mol. Endocrinol. 1997; 11: 183-192Crossref PubMed Scopus (50) Google Scholar,19Fabritz J. Ryan S. Ascoli M. Biochemistry. 1998; 37: 664-672Crossref PubMed Scopus (40) Google Scholar). Stably transfected cells were plated in 100-mm dishes and were metabolically labeled during a 4-h incubation in phosphate-free medium containing 200 μCi/ml32Pi. Receptor phosphorylation was ascertained after incubating the 32Pi-prelabeled cells at 37 °C with buffer only, or with 1000 ng/ml oLH for 5 min. These conditions were chosen to elicit a maximal response (5Hipkin R.W. Sánchez-Yagüe J. Ascoli M. Mol. Endocrinol. 1993; 7: 823-832Crossref PubMed Scopus (44) Google Scholar, 6Hipkin R.W. Wang Z. Ascoli M. Mol. Endocrinol. 1995; 9: 151-158Crossref PubMed Google Scholar, 8Wang Z. Hipkin R.W. Ascoli M. Mol. Endocrinol. 1996; 10: 748-759PubMed Google Scholar). One cell line expressing rLHR-wt (either 293L(wt-12) or 293L(wt-17)) and one cell line expressing one of the rLHR mutants was used in each experiment. Following lysis of the cells, the amount of wt and mutant receptor used for immunoprecipitation was equalized based on125I-hCG binding assays (see below). Immunoprecipitations were performed using Bugs (a rabbit polyclonal antibody to the rLHR (20Rosemblit N. Ascoli M. Segaloff D.L. Endocrinology. 1988; 123: 2284-2290Crossref PubMed Scopus (55) Google Scholar)) or a mixture of AntiL and R02 (two polyclonal antibodies directed against synthetic peptides derived from the known amino acid sequence of the rLHR (19Fabritz J. Ryan S. Ascoli M. Biochemistry. 1998; 37: 664-672Crossref PubMed Scopus (40) Google Scholar, 21Rodrı́guez M.C. Segaloff D.L. Endocrinology. 1990; 127: 674-681Crossref PubMed Scopus (40) Google Scholar)). The methodology used for immunoprecipitation and SDS gels was the same as that described earlier (5Hipkin R.W. Sánchez-Yagüe J. Ascoli M. Mol. Endocrinol. 1993; 7: 823-832Crossref PubMed Scopus (44) Google Scholar, 6Hipkin R.W. Wang Z. Ascoli M. Mol. Endocrinol. 1995; 9: 151-158Crossref PubMed Google Scholar, 8Wang Z. Hipkin R.W. Ascoli M. Mol. Endocrinol. 1996; 10: 748-759PubMed Google Scholar, 19Fabritz J. Ryan S. Ascoli M. Biochemistry. 1998; 37: 664-672Crossref PubMed Scopus (40) Google Scholar). Autoradiograms of the dried gels were obtained using Kodak BioMax MS film and intensifying screens. The autoradiograms were scanned using a Bio-Rad Molecular Imaging System and captured in a digital format for presentation. Equilibrium binding parameters for hCG were measured during an overnight incubation (4 °C) of intact cells with a fixed concentration of125I-hCG and increasing concentrations of hCG as described previously (6Hipkin R.W. Wang Z. Ascoli M. Mol. Endocrinol. 1995; 9: 151-158Crossref PubMed Google Scholar, 8Wang Z. Hipkin R.W. Ascoli M. Mol. Endocrinol. 1996; 10: 748-759PubMed Google Scholar). Concentration-response curves for the hCG-induced increases in cAMP accumulation were obtained by measuring total cAMP levels in cells that had been incubated with at least five different concentrations of hCG for 30 min at 37 °C in the presence of a phosphodiesterase inhibitor. The different parameters that describe the concentration response curves were calculated as described elsewhere (6Hipkin R.W. Wang Z. Ascoli M. Mol. Endocrinol. 1995; 9: 151-158Crossref PubMed Google Scholar, 8Wang Z. Hipkin R.W. Ascoli M. Mol. Endocrinol. 1996; 10: 748-759PubMed Google Scholar). Measurements of agonist-induced uncoupling in stably transfected cells were performed as follows. Cells expressing rLHR-wt or the mutant receptors were divided into two groups and incubated without (group A) or with (group B) 100 ng/ml oLH or hCG for 15 min. 3All data were combined because no differences were noted when oLH or hCG were used. All cells were then washed with neutral and acidic buffers to remove the free and bound hormone, respectively, and each group of cells was subdivided into two groups, which were then incubated without (groups A1 and B1) or with (groups A2 and B2) 100 ng/ml hCG for 15 min at 37 °C (6Hipkin R.W. Wang Z. Ascoli M. Mol. Endocrinol. 1995; 9: 151-158Crossref PubMed Google Scholar, 8Wang Z. Hipkin R.W. Ascoli M. Mol. Endocrinol. 1996; 10: 748-759PubMed Google Scholar). Intracellular levels of cAMP were measured at the end of this incubation, and agonist-induced uncoupling was calculated as follows: ((B2 − B1)/(A2 − A1)) × 100. This same assay could not be used to measure uncoupling in transiently transfected cells because of their reduced responsiveness. Thus, when using transiently transfected cells, uncoupling was simply assessed by measuring the total levels of cAMP accumulated by the cells during a 15-min incubation with a saturating concentration of hCG (100 ng/ml) in medium containing a phosphodiesterase inhibitor (6Hipkin R.W. Wang Z. Ascoli M. Mol. Endocrinol. 1995; 9: 151-158Crossref PubMed Google Scholar, 7Premont R.T. Macrae A.D. Stoffel R.H. Chung N. Pitcher J.A. Ambrose C. Inglese J. MacDonald M.E. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 6403-6410Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 8Wang Z. Hipkin R.W. Ascoli M. Mol. Endocrinol. 1996; 10: 748-759PubMed Google Scholar). The endocytosis of125I-hCG was measured in cells that had been briefly preincubated (i.e. 10 min at room temperature) with 40 ng/ml125I-hCG. At the end of this preincubation, the free hormone was removed by washing, and the cells were re-incubated in fresh, hormone-free medium at 37 °C for up to 4 h. After the desired interval, the cells were placed on ice, and the medium was saved. The cells were briefly treated with an isotonic pH 3 buffer (10Ascoli M. J. Biol. Chem. 1982; 257: 13306-13311Abstract Full Text PDF PubMed Google Scholar,13Dhanwada K.R. Vijapurkar U. Ascoli M. Mol. Endocrinol. 1996; 10: 544-554PubMed Google Scholar). The radioactivity that was released by the acid treatment was considered to be surface-bound, whereas the radioactivity that remained cell-associated was considered to be internalized. The medium was precipitated with trichloroacetic acid, and the acid-insoluble and -soluble radioactivity were considered to be undegraded and degraded hormone, respectively (10Ascoli M. J. Biol. Chem. 1982; 257: 13306-13311Abstract Full Text PDF PubMed Google Scholar, 13Dhanwada K.R. Vijapurkar U. Ascoli M. Mol. Endocrinol. 1996; 10: 544-554PubMed Google Scholar). Because there is little or no dissociation of the receptor-bound hCG during this incubation (10Ascoli M. J. Biol. Chem. 1982; 257: 13306-13311Abstract Full Text PDF PubMed Google Scholar), the rate of disappearance of the receptor bound hCG can be used to measure the rate of internalization. The rates of internalization (ke) were thus calculated from the slopes of linear regression fits to plots of the ln of the surface-bound hCGversus time. The half-life of internalization (t½) is therefore defined as 0.693/ke. Expression of the transfected arrestins and dynamin was ascertained by immunoblots using the ECL system of detection. The different arrestin constructs were detected using a polyclonal antibody (KEE) raised against a C-terminal 16 residue peptide (22Sterne-Marr R. Gurevich V.V. Goldsmith P. Bodine R.C. Sanders C. Donoso L.A. Benovic J.L. J. Biol. Chem. 1993; 268: 15640-15648Abstract Full Text PDF PubMed Google Scholar) or a mouse monoclonal antibody (F4C1) directed against an epitope common to all known arrestins (23Donoso L.A. Gregerson D.S. Smith L. Robertson S. Knospe V. Vrabec T. Kalsow C.M. Curr. Eye Res. 1990; 9: 343-355Crossref PubMed Scopus (51) Google Scholar). The HA-tagged dynamin was detected using the 12CA5 monoclonal antibody (Boehringer Mannheim). Purified hCG (CR-127) and oLH (AFP-5551B) were obtained from the National Hormone and Pituitary Agency of the NIDDK, National Institutes of Health.125I-hCG was prepared as described previously (24Ascoli M. Puett D. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 99-102Crossref PubMed Scopus (123) Google Scholar), to give a specific radioactivity of 25,000–30,000 cpm/ng. [32P]Orthophosphate was obtained from NEN Life Science Products. Phosphate-free DMEM was purchased from ICN Biomedicals (Irvine, CA). Nonidet P-40, protease inhibitors,N,N′,N“-triacetylchitotriose, protein A-agarose, and bovine serum albumin were from Sigma. Okadaic acid and cypermethrin were purchased from Alexis Biochemicals (Woburn, MA). Wheat germ agglutinin agarose was from Vector Laboratories. Cell culture supplies and reagents were obtained from Corning (Corning, NY) and Life Technologies, Inc., respectively. All other materials were obtained from commonly used suppliers. Phosphoamino acid analysis and phosphorylation experiments utilizing three different C-terminal truncations of the rLHR and a full-length mutant with multiple Ser to Ala mutations have identified Ser635, Ser639, Ser649, and Ser652 as the major locus of rLHR phosphorylation in transfected cells (6Hipkin R.W. Wang Z. Ascoli M. Mol. Endocrinol. 1995; 9: 151-158Crossref PubMed Google Scholar, 8Wang Z. Hipkin R.W. Ascoli M. Mol. Endocrinol. 1996; 10: 748-759PubMed Google Scholar, 9Wang Z. Liu X. Ascoli M. Mol. Endocrinol. 1997; 11: 183-192Crossref PubMed Scopus (50) Google Scholar). For the experiments presented here, we prepared four new full-length rLHR mutants in which each of these four serines was individually mutated to alanine. Each mutant cDNA (designated rLHR-S635A, rLHR-S639A, rLHR-S649A, and rLHR-S652A) was transfected into human kidney 293 cells, and clonal lines stably expressing each of the mutants were obtained. All mutant receptors bound hCG with an affinity comparable to that detected in cells expressing rLHR-wt (i.e. about 200 pm, see TableI). Of the several clonal lines obtained with each mutant, we chose those with the highest cell surface receptor density for further study (Table I).Table IhCG binding and cAMP responsiveness of clonal lines of 293 cells expressing the wild-type and mutant receptorsCell linehCG bindingaEquilibrium binding parameters were determined using intact cells during an overnight incubation at 4 °C as described under “Materials and Methods.” Each number represents the average ± S.E. of 4–6 independent experiments.cAMP responsivenessbTotal (i.e. cells + medium) levels of cAMP were measured in cells incubated in the presence of a phosphodiesterase inhibitor and increasing concentrations of hCG for 30 min or in cells incubated with a single concentration (500 ng/ml) of cholera toxin for 2 h, as described under “Materials and Methods.” The response ratio shown in the far right was calculated by dividing the average maximal response obtained for hCG over the average maximal response obtained for cholera toxin. Each number represents the average (±S.E.) of three independent experiments for the cell lines expressing the mutant receptors or nine independent experiments for the cell lines expressing rLHR-wt. The results obtained with 293L(wt-12) and 293L(wt-17) were combined because they seem to be indistinguishable.KdB maxBasalhCG responseCholera toxin responseResponse ratioEC50R maxR maxpmmolecules/cellpmol/106cellspmpmol/106 cellspmol/106cells293L(w-12) or 293L(wt-17)NDcND, not determined. These parameters were not remeasured in the current series of experiments, but previous data from this laboratory have shown that these two cell lines bind hCG with aKd of 200–300 pm and display aB max of 100,000–200,000 molecules/cell (8, 9,19).NDcND, not determined. These parameters were not remeasured in the current series of experiments, but previous data from this laboratory have shown that these two cell lines bind hCG with aKd of 200–300 pm and display aB max of 100,000–200,000 molecules/cell (8, 9,19).4.3 ± 0.739 ± 72678 ± 4891242 ± 1962.16293L(S635A-5)232 ± 31190,000 ± 17,0004.4 ± 1.270 ± 134541 ± 2201331 ± 683.41293L(S639A-5)170 ± 3178,000 ± 10,0002.6 ± 0.562 ± 212338 ± 775830 ± 1812.82293L(S649A-14)156 ± 13149,000 ± 26,0001.8 ± 0.475 ± 26538 ± 41330 ± 212.94293L(S652A-5)163 ± 20131,000 ± 18,0001.6 ± 0.486 ± 36744 ± 187360 ± 682.07a Equilibrium binding parameters were determined using intact cells during an overnight incubation at 4 °C as described under “Materials and Methods.” Each number represents the average ± S.E. of 4–6 independent experiments.b Total (i.e. cells + medium) levels of cAMP were measured in cells incubated in the presence of a phosphodiesterase inhibitor and increasing concentrations of hCG for 30 min or in cells incubated with a single concentration (500 ng/ml) of cholera toxin for 2 h, as described under “Materials and Methods.” The response ratio shown in the far right was calculated by dividing the average maximal response obtained for hCG over the average maximal response obtained for cholera toxin. Each number represents the average (±S.E.) of three independent experiments for the cell lines expressing the mutant receptors or nine independent experiments for the cell lines expressing rLHR-wt. The results obtained with 293L(wt-12) and 293L(wt-17) were combined because they seem to be indistinguishable.c ND, not determined. These parameters were not remeasured in the current series of experiments, but previous data from this laboratory have shown that these two cell lines bind hCG with aKd of 200–300 pm and display aB max of 100,000–200,000 molecules/cell (8Wang Z. Hipkin R.W. Ascoli M. Mol. Endocrinol. 1996; 10: 748-759PubMed Google Scholar, 9Wang Z. Liu X. Ascoli M. Mol. Endocrinol. 1997; 11: 183-192Crossref PubMed Scopus (50) Google Scholar,19Fabritz J. Ryan S. Ascoli M. Biochemistry. 1998; 37: 664-672Crossref PubMed Scopus (40) Google Scholar). Open table in a new tab The data presented in Table I also show a comparison of the concentration response curves for hCG-induced cAMP accumulation in the clonal lines expressing the different mutants and in two clonal lines, 293L(wt-12) and 293L(wt-17), stably transfected with the rLHR-wt. We chose 293L(wt-12) and 293L(wt-17) as control cell lines because their receptor density (100,000-200,000 receptors/cell) is comparable to that of the cell lines expressing the mutant receptors, and at these levels of receptor expression, the cAMP response is basically independent of receptor density (6Hipkin R.W. Wang Z. Ascoli M. Mol. Endocrinol. 1995; 9: 151-158Crossref PubMed Google Scholar, 8Wang Z. Hipkin R.W. Ascoli M. Mol. Endocrinol. 1996; 10: 748-759PubMed Google Scholar, 19Fabritz J. Ryan S. Ascoli M. Biochemistry. 1998; 37: 664-672Crossref PubMed Scopus (40) Google Scholar). In an attempt to correct for an inherent variability in the basal and hCG-stimulated levels of cAMP, we also measured the levels of cAMP in the different cell lines stimulated with cholera toxin and calculated a response ratio by dividing the maximal hCG response by the maximal cholera toxin response. As shown in TableI, this ratio shows that all mutants respond to hCG as well as or better than cells expressing rLHR-wt. It is noted" @default.
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• W2056528957 title "Mutation of Individual Serine Residues in the C-terminal Tail of the Lutropin/Choriogonadotropin Receptor Reveal Distinct Structural Requirements for Agonist-induced Uncoupling and Agonist-induced Internalization" @default.
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