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Gonadotropin-Releasing Hormone Receptor Concentration Differentially Regulates Intracellular Signaling Pathways in GGH3 Cells

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Abstract

Pituitary cells line (GGH3) expressing the GnRH receptor (GnRHR) were used to investigate the effect of GnRHR concentration on the ability of a GnRH agonist to activate second messenger systems. Four different strategies were utilized to generate cells expressing functionally different concentrations of receptors: (1) transient transfection with different concentrations of wild type GnRHR into GH3 cells, (2) utilization of two cell lines derived from a common stably transfected line expressing high (4,209 ± 535 receptors/cell) or low (1,031 ± 36 receptors/cell) concentrations of GnRHR, (3) co-incubation of GGH3-1′ cells with a GnRH agonist (Buserelin) and a GnRH antagonist to compete for binding sites, and (4) photo-affinity binding to GnRHR with a GnRH antagonist to change effective receptor concentration. A range of receptor concentrations (1,000–8,000 receptors/cell) were generated by these techniques. Inositol phosphate (IP) and cAMP accumulation were quantified to assess the effect of receptor concentration on receptor-effector coupling. Under all four paradigms, the efficacy and potency of Buserelin stimulated IP production was dependent on receptor concentration. In contrast, Buserelin stimulated cAMP release was relatively unchanged at varying concentrations of GnRHR. This suggests that the cellular concentration of GnRHR affects the induction of cell signaling pathways. These results demonstrate that a single ligand-receptor-complex can differentially activate second messenger systems and present a mechanism by which multiple physiological endpoints can be differentially regulated by a single hormone/receptor interaction.

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References

  1. Marian J, Cooper RL, Conn PM. Regulation of the rat gonadotropin-releasing hormone receptor. Mol Pharm 1981; 19:399–405.

    Google Scholar 

  2. Clayton RN. Gonadotrophin-releasing hormone: Its actions and receptors. Endocrinology 1989;120:11–19.

    Google Scholar 

  3. Dalkin AC, Haisenleder DJ, Ortolano GA, Ellis TR, Marshall JC. The frequency of gonadotropin-releasing-hormone stimulation differentially regulates gonadotropin subunit messenger ribonucleic acid expression. Endocrinology 1989; 125:917–924.

    Google Scholar 

  4. Conn PM, Janovick JA, Braden TD, Maurer RA, Jennes L. SIIp: A unique secretogranin/chromogranin of the pituitary released in response to gonadotropin-releasing hormone. Endocrinology 1992;130:3033–3040.

    Google Scholar 

  5. Janovick JA, Jennes L, Conn PM. GH3 cells transfected with gonadotropin-releasing hormone (GnRH) receptor complementary deoxyribonucleic acid release secretogranin-II through a constitutive pathway after GnRH analogregulated synthesis: Evidence that secretory proteins do not contain a sequence that obligates processing through a secretory granule or by regulated secretion. Endocrinology 1995;136:202–208.

    Google Scholar 

  6. Conn PM, Janovick JA, Stanislaus D, Kuphal D, Jennes L. Molecular and cellular basis of gonadotropin-releasing hormone action in the pituitary and central nervous system. Vit Horm 1995;50:151–214.

    Google Scholar 

  7. Kaiser UB, Conn PM, Chin WW. 1997 Studies of gonadotropin-releasing hormone (GnRH) action using GnRH receptor-expressing pituitary cell lines. Endocr Rev 1997;18: 46–70.

    Google Scholar 

  8. Stojilkovic SS. Calcium signaling systems. In: Conn PM(ed) Handbook of Physiology: Molecular Endocrinology. London: Oxford Press, 1998.

    Google Scholar 

  9. Hille BL, Tse A, Tse FW, Bosma MM. Signaling mechanisms during the response of pituitary gonadotropes to GnRH. Recent Prog Horm Res 1995;50:75–85.

    Google Scholar 

  10. Hawes BE, Marzen JE, Waters SB, Conn PM. Sodium fluoride provokes gonadotrope desensitization to gonadotropin-releasing hormone (GnRH) and gonadotrope sensitization to A23187: Evidence for multiple G-proteins in GnRH action. Endocrinology 1992;130:2465–2475.

    Google Scholar 

  11. Hawes BE, Barnes S, Conn PM. Cholera toxin and pertussis toxin provoke differential effects on luteinizing hormone release, inositol phosphate production, and gonadotropin releasing hormone receptor binding in the gonadotrope: Evidence for multiple guanyl nucleotide binding proteins in GnRH action. Endocrinology 1993;132:2124–2130.

    Google Scholar 

  12. Hsieh KP, Martin TFJ. Thyrotropin-releasing hormone and gonadotropin-releasing hormone receptors activate phospholipase C by coupling to the guanosine triphosphate-binding proteins Gq and G11. Mol Endocrinol 1992;6:1673–1681.

    Google Scholar 

  13. Stanislaus D, Janovick JA, Brothers S, Conn PM. Regulation of Gq/11a by the GnRH receptor. Mol Endocrinol 1997; 11:738–746.

    Google Scholar 

  14. Cornea A, Janovick JA, Stanislaus D, Conn PM. Redistribution of Gq/11a in the pituitary gonadotrope in response to a GnRH agonist. Endocrinology 1998;139:397–402.

    Google Scholar 

  15. Stanislaus D, Ponder S, Ji TH, Conn PM. GnRH receptor coupling to multiple G-proteins in gonadotropes and GGH3 cells: Evidence from palmitoylation and overexpression of G-proteins. Biology of Reproduction 1998;59:579–586.

    Google Scholar 

  16. Stanislaus D, Janovick JA, Jennes L, Kaiser UB, Chin WW, Conn PM. Functional and morphological characterization of four cell lines derived from GH3 cells stably transfected with gonadotropin-releasing hormone receptor complementary deoxyribonucleic acid. Endocrinology 1994;135:2220–2227.

    Google Scholar 

  17. Imai A, Takagi H, Horibe S, Fuseya T, Tamaya T. Coupling of gonadotropin-releasing hormone receptor to Gi protein in human reproductive tract tumors. J Clin Endocrinol Metab 1996;81:3249–3253.

    Google Scholar 

  18. Cotecchia S, Kobilka BK, Daniel KW, Nolan RD, Lapetina EY, Caron MG, Lefkowitz RJ, Regan JW. Multiple second messenger pathways of a-adrenergic receptor subtypes expressed in eukaryotic cells. J Biol Chem 1990;265:63–69.

    Google Scholar 

  19. Valler L, Muca C, Magni M, Albert P, Bunzow J, Meldolesi J, Civelli O. Differential coupling of dopaminergic D2 receptors expressed in different cell types. Stimulation of phosphatidyl 4,5-bisphosphate hydrolysis in LtK fibroblasts, hyperpolarization, and cytosolic-free Ca22 concentration decrease in GH4C1 cells. J Biol Chem 1990;265:10320–20326.

    Google Scholar 

  20. Perez DM, Deyoung MB, Graham RM. Coupling of expressed a1B and a1D-adrenergic receptor to multiple signaling pathways is both G-protein and cell type specific. Mol Pharmacol 1993;44:784–795.

    Google Scholar 

  21. Herzog H, Hort YJ, Ball HJ, Hayes G, Shine J, Selbie LA. Cloned human neuropeptide Y receptor couples to 2 different second messenger systems. Proc Natl Acad Sci USA 1992;89:5794–5798.

    Google Scholar 

  22. Huang C, Hepler JR, Chen LT, Gilman AG, Anderson RGW, Mumby SM. Organization of G-proteins and adenylyl cyclase at the plasma membrane. Mol Biol Cell 1997;8:2365–2378.

    Google Scholar 

  23. Katt JA, Duncan JA, Herbon L, Barkan A, Marshall JC. The frequency of gonadotropin-releasing hormone stimulation determines the number of pituitary gonadotropin-releasing hormone receptors. Endocrinology 1985;116:2113–2115.

    Google Scholar 

  24. Kaiser UB, Jakubowiak a, Steinberger A, Chin WW. Regulation of rat pituitary gonadotropin-releasing hormone receptor mRNA levels in vivo and in vitro. Endocrinology 1993;133:931–934.

    Google Scholar 

  25. Yasin M, Dalkin AC, Haisenleder DJ, Marshall JC. GnRH pulse pattern regulates GnRH receptor gene expression: Augmentation by estradiol. Endocrinology 1995;136:1559–1564.

    Google Scholar 

  26. Kaiser UB, Sabbagh B, Katzenellenbogen RA, Conn PM, Chin WW. A mechanism for the differential regulation of gonadotropin subunit gene expression by gonadotropin-releasing hormone. Proc Natl Acad Sci USA 1995;92:12280–12284.

    Google Scholar 

  27. Selbie LA, Hill SJ. G protein-coupled-receptor cross-talk: The fine tuning of multiple receptor-signaling pathways. Trends Pharmacol Sci 1998;19:87–93.

    Google Scholar 

  28. Kupjal D, Janovick JA, Kaiser UB, Chin WW, Conn PM. Stable transfection of GH3 cells with rat gonadotropin releasing hormone receptor complementary deoxyribonucleic acid results in expression of a receptor coupled to cyclic adenosine 39,59-monophosphate-dependent prolactin release via a G-protein. Endocrinology 1994;135:315–320.

    Google Scholar 

  29. Haviv F, Fitzpatrick TD, Nochols CJ, Swenson RE, Mort NA, Bush EN, Diaz G, Nguyen AT, Holst MR, Cybulski VA, Leal JA, Bammert G, Rhutasel NS, Dodge PW, Johnson ES, Cannon JB, Knittle J, Greer J. The effect of NMeTyr5 substitution in luteinizing hormone-releasing hormone antagonists. J Med Chem 1993;36:928–933.

    Google Scholar 

  30. Janovick JA, Haviv F, Fitzpatrick TD, Conn PM. Differential orientation of a GnRH agonist and antagonist in the pituitary GnRH receptor. Endocrinology 1993;133:942–945.

    Google Scholar 

  31. McArdle CA, Gorospe WC, Huckle WR, Conn PM. Homologous down-regulation of gonadotropin-releasing hormone receptors and desensitization of gonadotropes: Lack of dependence on protein kinase C. Mol Endocrinol 1987;1:420–429.

    Google Scholar 

  32. Huckle WR, Conn PM. The relationship between gonadotropin-releasing hormone-stimulated luteinizing hormone release and inositol phosphate production: Studies with calcium antagonists and protein kinase C activators. Endocrinology 1987;120:160–169.

    Google Scholar 

  33. Steiner AL, Perker CW, Kipnis DM. Radioimmunoassay for cyclic nucleotides. J Biol Chem 1972;247:1106–1113.

    Google Scholar 

  34. Harper JF, Brooker G. Femtomole sensitive radioimmunoassay of cyclic AMP and cyclic GMP after 290 acetylation by acetic anhydride in aqueous solution. J Cyclic Nucleotide Res 1975;1:207–218.

    Google Scholar 

  35. Andrews WV, Staley DD, Huckle WR, Conn PM. Stimulation of luteinizing hormone (LH) release and phospholipid breakdown by guanosine triphosphate in permeabilized pituitary gonadotropes: Antagonist action suggests association of a G protein and gonadotropin-releasing hormone receptor. Endocrinology 1986;119:2537–2546.

    Google Scholar 

  36. Tsuda T, Kusui T, HouW, Benya RV, Akeson MA, Kroog GS, Battey JF, Jensen RT. Affect of gastrin-releasing peptide receptor number on receptor affinity, coupling, degradation, and modulation. Mol Pharm 1997;51:721–732.

    Google Scholar 

  37. Zhu X, Gilbert S, Birnbaumer M, Birnbaumer L. Dual signaling potential is common among Gs-coupled receptors and dependent on receptor density. Mol Pharmacol 1994;46: 460–469.

    Google Scholar 

  38. Guo J, Iida-Klein A, Huang X, Abou-Samra AB, Segre GV, Bringhurst FR. Parathyroid hormone (PTH)/PTH-related peptide receptor density modulates activation of phospholipase C and phosphate transport by PTH in LLC-PK1 cells. Endocrinology 1995;136:3884–3891.

    Google Scholar 

  39. Gilchrist RL, Ryu K-S, Ji I, Ji TH. The luteinizing hormone/ chorionic gonadotropin receptor has distinct transmembrane conductors for cAMP and inositol phosphate signals. J Biol Chem 1996; 271:19283–19287.

    Google Scholar 

  40. Barnes SJ, Conn PM. Cholera toxin and dibutyryl cyclic adenosine 39,59-monophosphate sensitize gonadotropin-releasing hormone-stimulated inositol phosphate production to inhibition in protein kinase-C (PKC)-depleted cells: Evidence for cross-talk between a cholera toxin-sensitive Gprotein and PKC. Endocrinology 1993;133:2756–2760.

    Google Scholar 

  41. Laglia G. Increased cyclic adenosine 39,59-monophosphate inhibits G protein-coupled activation of phospholipase C in rat FRTL-5 thyroid cells. Endocrinology 1996;137:3170–3176.

    Google Scholar 

  42. Abdel-Latiff AA. Cross talk between cyclic AMP and the polyphosphoinositide signaling cascade in iris sphincter and other novascular smooth muscle. Proc Soc Exp Biol Med 1996;211:163–177.

    Google Scholar 

  43. Tang WJ, Gilman AG. Type-specific regulation of adenylyl cyclase by G protein beta gamma subunits. Science 1991;254: 1500–1503.

    Google Scholar 

  44. Federman AD, Conklin BR, Schrader KA, Reed RR, Bourne HR. Hormonal stimulation of adenylyl cyclase through Giprotein beta gamma subunits. Nature 1992;356:159–161.

    Google Scholar 

  45. Pian MS, Dobbs LG. Evidence for G beta gamma-mediated cross-talk in primary cultures of lung alveolar cells. Pertussis toxin-sensitive production of cAMP. J Biol Chem 1995; 270:7427–7430.

    Google Scholar 

  46. Spence S, Rena G, Sweeney G, Houslay MD. Induction of Ca21 /calmodulin-stimulated cyclic AMP phosphodiesterase (PDE1) activity in Chinese hamster ovary (CHO) by phorbol 12-myristate 13-acetate and by the selective overexpression of protein kinase C isoforms. Biochem J 1995;310:975–982.

    Google Scholar 

  47. Garrel G, McArdle CA, Hemmings BA, Counis R. Gonadotropin-releasing hormone and pituitary adenylate cyclaseactivating polypeptide affect levels of cyclic adenosine 39,59-monophosphate-dependent protein kinase A (PKA) subunits in the clonal gonadotrope alphaT3–1 cells: Evidence for cross-talk between PKA and protein kinase C pathways. Endocrinology 1997;138:2259–2266.

    Google Scholar 

  48. Cornea A, Janovick JA, Lin X, Conn PM. Simultaneous and independent visualization of the gonadotropin releasing hormone receptor and its ligand: Evidence for independent processing and recycling on living cells. Endocrinology 1999;140:4272–4280.

    Google Scholar 

  49. Lin X, Janovick JA, Brothers S, Blomenrohr M, Bogerd J, Conn PM. Addition of catfish gonadotropin-releasing hormone (GnRH) receptor intracellular carboxyl-terminal tail to rat GnRH receptor alters receptor expression and regulation. Mol Endocrinol 1998;12:161–171.

    Google Scholar 

  50. Gubitz AK, Widdowson L, Kurokawa M, Kirkpatrick KA, Richardson PJ. Dual signaling by the adenosine A2a receptor involves activation of both N-and P-type calcium channels by different G-proteins and protein kinases in the same striatal nerve terminals. J Neurochem 1996;67:374–381.

    Google Scholar 

  51. Herrlich A, Kuhn B, Grosse R, Schmid A, Schultz G, Gudermann T. Involvement of Gs and Gi proteins in dual coupling of the luteinizing hormone receptor to adenylyl cyclase and phospholipase C. J Bio Chem 1996;271:16764–16772.

    Google Scholar 

  52. Ashkenazi A, Winslow JW, Peralta EG, Peterson GL, Schimerlik MI, Capon DJ, Ramachandran J. An M2 muscarinic receptor subtype coupled to both adenylyl cyclase and phosphoinositide turnover. Science 1987;238:672–675.

    Google Scholar 

  53. Offermans S, Simon MI. Organization of transmembrane signaling by heterotrimeric G-proteins. Cancer Surveys 1996;27:177–198.

    Google Scholar 

  54. Conn PM, Rogers DC, Stewart JM, Neidel J, Sheffield T. Conversion of a gonadotropin releasing hormone antagonist to an agonist: Implication for a receptor microaggregate as the functional unit for signal transduction. Nature 1982; 296:653–655.

    Google Scholar 

  55. Blum JJ, Conn PM. Gonadotropin-releasing hormone stimulation of luteinizing hormone: A ligand-receptor-effector model. Proc Natl Acad Sci USA 1982;79:7307–7311.

    Google Scholar 

  56. Jennes L, Bronson D, Stumpf WR, Conn PM. Evidence for an association between calmodulin and membrane patches containing gonadotropin-releasing hormone-receptor complexes in cultured gonadotropes. Cell Tissue Res 1985;239: 311–315.

    Google Scholar 

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Authors and Affiliations

  1. Divisions of Neuroscience and Reproductive Biology, Oregon Regional Primate Research Center, Beaverton, Oregon

    Jonathon H. Pinter, Jo Ann Janovick & P. Michael Conn

  2. Department of Physiology and Pharmacology, Oregon Health Sciences University, Portland, Oregon

    P. Michael Conn

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Pinter, J.H., Janovick, J.A. & Conn, P.M. Gonadotropin-Releasing Hormone Receptor Concentration Differentially Regulates Intracellular Signaling Pathways in GGH3 Cells. Pituitary 2, 181–190 (1999). https://doi.org/10.1023/A:1009946807430

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