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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1980 Jun;77(6):3440–3444. doi: 10.1073/pnas.77.6.3440

Aggregation of luteinizing hormone receptors in granulosa cells: a possible mechanism of desensitization to the hormone.

A Amsterdam, A Berkowitz, A Nimrod, F Kohen
PMCID: PMC349632  PMID: 6251459

Abstract

The temporal relationship between redistribution of receptors to lutropin (luteinizing hormone)/human chorionic gonadotropin in cultured rat ovarian granulosa cells and the cellular response to hormonal challenge were studied. Visualization of receptor-bound human chorionic gonadotropin by indirect immunofluorescence, with hormone-specific antibodies after fixation with 2% formaldehyde, revealed the existence of small clusters around the entire cell circumference 5--20 min after exposure to the hormone at 37 degrees C. Such small receptor aggregates were also evident if hormone incubation was at 4 degrees C or if cells were fixed with 2% formaldehyde before incubation. Larger clusters were evident after prolonged incubation with the hormone (2--4 hr) at 37 degrees C. The later change coincided with diminished cyclic AMP accumulation in respose to challenge with fresh hormone. When the fixation step was omitted and antibodies to human chorionic gonadotropin were applied after hormonal binding, acceleration of both receptor clustering and the desensitization process was observed. This maneuver also induced capping of the hormone receptors. In contrast, monovalent Fab' fragments of the antibodies were without effect. Internalization of the bound hormone in lysosomes, and subsequent degradation, was evident 8 hr after hormonal application and was not accelerated by the antibodies. It is suggested that clustering of the luteinizing hormone receptors may play a role in cellular responsiveness to the hormone. Massive aggregation of the receptors may desensitize the cell by interferring with coupling to adenylate cyclase.

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Selected References

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  1. Amsterdam A., Nimrod A., Lamprecht S. A., Burstein Y., Lindner H. R. Internalization and degradation of receptor-bound hCG in granulosa cell cultures. Am J Physiol. 1979 Feb;236(2):E129–E138. doi: 10.1152/ajpendo.1979.236.2.E129. [DOI] [PubMed] [Google Scholar]
  2. Brown M. S., Goldstein J. L. Receptor-mediated control of cholesterol metabolism. Science. 1976 Jan 16;191(4223):150–154. doi: 10.1126/science.174194. [DOI] [PubMed] [Google Scholar]
  3. Carpenter G., Cohen S. 125I-labeled human epidermal growth factor. Binding, internalization, and degradation in human fibroblasts. J Cell Biol. 1976 Oct;71(1):159–171. doi: 10.1083/jcb.71.1.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Edelman G. M. Surface modulation in cell recognition and cell growth. Science. 1976 Apr 16;192(4236):218–226. doi: 10.1126/science.769162. [DOI] [PubMed] [Google Scholar]
  5. Fraker P. J., Speck J. C., Jr Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphrenylglycoluril. Biochem Biophys Res Commun. 1978 Feb 28;80(4):849–857. doi: 10.1016/0006-291x(78)91322-0. [DOI] [PubMed] [Google Scholar]
  6. Frye L. D., Edidin M. The rapid intermixing of cell surface antigens after formation of mouse-human heterokaryons. J Cell Sci. 1970 Sep;7(2):319–335. doi: 10.1242/jcs.7.2.319. [DOI] [PubMed] [Google Scholar]
  7. Gilman A. G. A protein binding assay for adenosine 3':5'-cyclic monophosphate. Proc Natl Acad Sci U S A. 1970 Sep;67(1):305–312. doi: 10.1073/pnas.67.1.305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Han S. S., Rajaniemi H. J., Cho M. I., Hirshfield A. N., Midgley A. R., Jr Gonadotropin receptors in rat ovarian tissue. II. Subcellular localization of LH binding sites by electron microscopic radioautography. Endocrinology. 1974 Aug;95(2):589–598. doi: 10.1210/endo-95-2-589. [DOI] [PubMed] [Google Scholar]
  9. Hanski E., Rimon G., Levitzki A. Adenylate cyclase activation by the beta-adrenergic receptors as a diffusion-controlled process. Biochemistry. 1979 Mar 6;18(5):846–853. doi: 10.1021/bi00572a017. [DOI] [PubMed] [Google Scholar]
  10. Harden T. K., Su Y. F., Perkins J. P. Catecholamine-induced desensitization involves an uncoupling of beta-adrenergic receptors and adenylate cyclase. J Cyclic Nucleotide Res. 1979;5(2):99–106. [PubMed] [Google Scholar]
  11. Harwood J. P., Conti M., Conn P. M., Dufau M. L., Catt K. J. Receptor regulation and target cell responses: studies in the ovarian luteal cell. Mol Cell Endocrinol. 1978 Jul-Aug;11(2):121–135. doi: 10.1016/0303-7207(78)90001-1. [DOI] [PubMed] [Google Scholar]
  12. Hirata F., Strittmatter W. J., Axelrod J. beta-Adrenergic receptor agonists increase phospholipid methylation, membrane fluidity, and beta-adrenergic receptor-adenylate cyclase coupling. Proc Natl Acad Sci U S A. 1979 Jan;76(1):368–372. doi: 10.1073/pnas.76.1.368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hoffman B. B., Mullikin-Kilpatrick D., Lefkowitz R. J. Desensitization of beta-adrenergic stimulated adenylate cyclase in turkey erythrocytes. J Cyclic Nucleotide Res. 1979 Oct;5(5):355–366. [PubMed] [Google Scholar]
  14. Humes J. L., Rounbehler M., Kuehl F. A., Jr A new assay for measuring adenyl cyclase activity in intact cells. Anal Biochem. 1969 Nov;32(2):210–217. doi: 10.1016/0003-2697(69)90077-3. [DOI] [PubMed] [Google Scholar]
  15. Kahn C. R., Baird K. L., Jarrett D. B., Flier J. S. Direct demonstration that receptor crosslinking or aggregation is important in insulin action. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4209–4213. doi: 10.1073/pnas.75.9.4209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lamprecht S. A., Zor U., Salomon Y., Koch Y., Ahren K., Lindner H. R. Mechanism of hormonally induced refractoriness of ovarian adenylate cyclase to luteinizing hormone and prostaglandin E. J Cyclic Nucleotide Res. 1977 Apr;3(2):69–83. [PubMed] [Google Scholar]
  17. Lamprecht S. A., Zor U., Tsafriri A., Lindner H. R. Action of prostaglandin E 2 and of luteinizing hormone on ovarian adenylate cyclase, protein kinase and ornithine decarboxylase activity during postnatal development and maturity in the rat. J Endocrinol. 1973 May;57(2):217–233. doi: 10.1677/joe.0.0570217. [DOI] [PubMed] [Google Scholar]
  18. Marchalonis J. J. An enzymic method for the trace iodination of immunoglobulins and other proteins. Biochem J. 1969 Jun;113(2):299–305. doi: 10.1042/bj1130299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Marsh J. M., Mills T. M., Lemaire W. J. Preovulatory changes in the synthesis of cyclic AMP by rabbit Graafian follicles. Biochim Biophys Acta. 1973 Mar 30;304(1):197–202. doi: 10.1016/0304-4165(73)90128-1. [DOI] [PubMed] [Google Scholar]
  20. NISONOFF A., MARKUS G., WISSLER F. C. Separation of univalent fragments of rabbit antibody by reduction of a single, labile disulphide bond. Nature. 1961 Jan 28;189:293–295. doi: 10.1038/189293a0. [DOI] [PubMed] [Google Scholar]
  21. Nimrod A., Erickson G. F., Ryan K. J. A specific FSH receptor in rat granulosa cells: properties of binding in vitro. Endocrinology. 1976 Jan;98(1):56–64. doi: 10.1210/endo-98-1-56. [DOI] [PubMed] [Google Scholar]
  22. Nimrod A., Lindner H. R. A synergistic effect of androgen on the stimulation of progesterone secretion by FSH in cultured rat granulosa cells. Mol Cell Endocrinol. 1976 Aug-Sep;5(3-4):315–320. doi: 10.1016/0303-7207(76)90093-9. [DOI] [PubMed] [Google Scholar]
  23. Orly J., Schramm M. Coupling of catecholamine receptor from one cell with adenylate cyclase from another cell by cell fusion. Proc Natl Acad Sci U S A. 1976 Dec;73(12):4410–4414. doi: 10.1073/pnas.73.12.4410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Orly J., Schramm M. Fatty acids as modulators of membrane functions: catecholamine-activated adenylate cyclase of the turkey erythrocyte. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3433–3437. doi: 10.1073/pnas.72.9.3433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Prives J., Silman I., Amsterdam A. Appearance and disappearance of acetycholine receptor during differentiation of chick skeletal muscle in vitro. Cell. 1976 Apr;7(4):543–550. doi: 10.1016/0092-8674(76)90204-x. [DOI] [PubMed] [Google Scholar]
  26. Salomon Y., Yanovsky A., Mintz Y., Amir Y., Lindner H. R. Synchronous generation of ovarian hCG binding sites and LH-sensitive adenylate cyclase in immature rats following treatment with pregnant mare serum gonadotropin. J Cyclic Nucleotide Res. 1977 Jun;3(3):163–176. [PubMed] [Google Scholar]
  27. Schlegel W., Kempner E. S., Rodbell M. Activation of adenylate cyclase in hepatic membranes involves interactions of the catalytic unit with multimeric complexes of regulatory proteins. J Biol Chem. 1979 Jun 25;254(12):5168–5176. [PubMed] [Google Scholar]
  28. Schlessinger J., Shechter Y., Cuatrecasas P., Willingham M. C., Pastan I. Quantitative determination of the lateral diffusion coefficients of the hormone-receptor complexes of insulin and epidermal growth factor on the plasma membrane of cultured fibroblasts. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5353–5357. doi: 10.1073/pnas.75.11.5353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Selstam G., Rosberg S., Liljekvist J., Grönquist L., Perklev T., Ahrén K. Differences in action of LH and FSH on the formation of cyclic AMP in the prepubertal rat ovary. Acta Endocrinol (Copenh) 1976 Jan;81(1):150–164. doi: 10.1530/acta.0.0810150. [DOI] [PubMed] [Google Scholar]
  30. Tokuyasu K. T., Singer S. J. Improved procedures for immunoferritin labeling of ultrathin frozen sections. J Cell Biol. 1976 Dec;71(3):894–906. doi: 10.1083/jcb.71.3.894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Zor U., Lamprecht S. A., Misulovin Z., Koch Y., Lindner H. R. Refractoriness of ovarian adenylate cyclase to continued hormonal stimulation. Biochim Biophys Acta. 1976 May 28;428(3):761–765. doi: 10.1016/0304-4165(76)90206-3. [DOI] [PubMed] [Google Scholar]
  32. de Petris S., Raff M. C. Distribution of immunoglobulin on the surface of mouse lymphoid cells as determined by immunoferritin electron microscopy. Antibody-induced, temperature-dependent redistribution and its implications for membrane structure. Eur J Immunol. 1972 Dec;2(6):523–535. doi: 10.1002/eji.1830020611. [DOI] [PubMed] [Google Scholar]

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