Abstract
Consecutive challenges with thyrotropin-releasing hormone (TRH) of oocytes expressing the TRH receptor (TRH-R) resulted in a pronounced desensitization, manifested as a decrease in chloride current amplitude and an increase in response latency. Exposure to low concentrations of TRH resulted in a marked decrease in the amplitude of the subsequent response to a higher concentration of the agonist, even though the second challenge was given before the onset of the response to the first challenge (within 3–15 s). Cellular calcium concentration ([Ca]i) did not increase within this interval, suggesting that calcium was not involved in the desensitization process. The latency of the second response, however, was either unchanged or shortened, implying additive effects of processes initiated by the first challenge. A longer interval (30 s) between the two challenges brought about a more pronounced decrease in amplitude and a prolongation of response latency. The calcium mobilization initiated by a second challenge with a high concentration of the agonist exhibited a longer latency, a lower rate of [Ca]i increase and a lower amplitude. Stimulation of coexpressed cholinergic-muscarinic ml receptors with a low concentration of acetylcholine resulted in a pronounced desensitization of the TRH response (heterologous desensitization). Activation of protein kinase C by β-phorbol 12-myristate,13-acetate resulted in a dose-dependent inhibition of the response to TRH, suggesting that protein kinase C was involved in desensitization. Chelerythrine, a specific inhibitor of protein kinase C, abolished a large part of the desensitization. A mutant of the TRH-R that lacks protein kinase C concensus phosphorylation sites in the C-terminal region, exhibited desensitization. Hence, desensitization is not targeted at this part of the receptor molecule. Our results suggest that a very low receptor occupancy activates an amplification step that results in heterologous desensitization. This process is mediated, at least partly, by the activation of protein kinase C, acting on a target proximal to calcium mobilization.
Similar content being viewed by others
References
Aizawa T, Hinkle PM (1985) Differential effects of thyrotropin-releasing hormone, vasoactive intestinal peptide, phorbol ester, and depolarization in GH4C1 rat pituitary cells. Endocrinology 116:909–919
Bennett CF, Crooke ST (1987) Purification and characterization of a phosphoinositide-specific phospholipase C from guinea pig uterus. J Biol Chem 262:13789–13797
Benovic JL, Strasser RH, Caron MG, Lefkowitz RJ (1986) β-Adrenergic receptor kinase: identification of a novel protein kinase that phosphorylates the agonist-occupied form of the receptor. Proc Natl Acad Sci USA 83:2797–2801
Carlson KE, Brass LF, Manning DR (1989) Thrombin and phorbol esters cause the selective phosphorylation of a protein other than Gi in human platelets. J Biol Chem 264:13298–13305
Clark RB, Kunkel MW, Friedman J, Goka TJ, Johnson JA (1988) Activation of cAMP-dependent protein kinase is required for heterologous desensitization of adenylyl cyclase in S49 wild-type lymphoma cells. Proc Natl Acad Sci USA 85:1442–1446
Connolly TM, Lawing WJ Jr, Majerus PW (1986) Protein kinase C phosphorylates human platelet inositol trisphosphate 5′-phosphomonoesterase, increasing the phosphatase activity. Cell 46:951–958
Dascal N, Landau EM, Lass Y (1984) Xenopus oocytes resting potential, muscarinic responses and the role of calcium and guanosine 3′,5′-cyclic monophosphate. J Physiol (Lond) 352:551–574
Dascal N, Gillo B, Lass Y (1985) Role of calcium mobilization in mediation of acetylcholine-evoked chloride currents in Xenopus laevis oocytes. J Physiol (Lond) 366:299–313
Gautvik MK, Gordeladze JO, Jahnsen T, Haug E, Hansson V, Lystad E (1983) Thyroliberin receptor binding and adenylyl cyclase activation in cultured prolactin-producing rat pituitary tumor cells (GH cells). J Biol Chem 258:10304–10311
Gillo B, Lass Y, Nadler E, Oron Y (1987) The involvement of inositol 1,4,5-trisphosphate and calcium in the two-component response to acetylcholine in Xenopus oocytes. J Physiol (Lond) 392:349–361
Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of calcium indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450
Haga K, Haga T (1992) Activation by G-protein βγ subunits of agonistor light-dependent phosphorylation of muscarinic acetylcholine receptors and rhodopsin. J Biol Chem 267: 2222–2227
Herbert JM, Augereau JM, Gleye J, Maffrand JP (1990) Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochim Biophys Acta 172:993–999
Kameyama K, Haga K, Haga T, Kontani K, Katade T, Fukada Y (1993) Activation by G protein βγ subunits of β-adrenergic and muscarinic receptor kinase. J Biol Chem 268:7753–7758
Kwatra MM, Benovic JL, Caron MG, Lefkowitz RJ, Hosey M (1989) Phosphorylation of chick heart muscarinic cholinergic receptors by the beta adrenergic receptor kinase. Biochemistry 28:4543–4547
Larsson C, Simonsson P (1993) Desensitization of acetylcholine-induced inositol 1,4,5-trisphosphate formation in neuroblastoma SH-SY5Y cells following repetitive acetylcholine stimulation. Neurosci Lett 150:141–144
Lipinsky D, Gershengorn MC, Oron Y (1993) Latency in the inositol lipid transduction pathway: the role of cellular events in responses to thyrotropin-releasing hormone in Xenopus oocytes. Pflügers Arch 425:140–149
Lohse JM (1993) Molecular mechanisms of membrane receptor desensitization. Biochim Biophys Acta 1179:171–188
Lupu-Meiri M, Shapira H, Oron Y (1989) Dual regulation by protein kinase C of the muscarinic response in Xenopus oocytes. Pflügers Arch 413:498–504
Lupu-Meiri M, Shapira H, Matus-Leibovitch N, Oron Y (1990) Two types of intrinsic muscarinic responses in Xenopus oocytes: I-differences in latencies and 45Ca efflux kinetics. Pflügers Arch 417:391–397
Lupu-Meiri M, Beit-Or A, Christensen SB, Oron Y (1993) Calcium entry in Xenopus oocytes: effects of inositol trisphosphate, thapsigargin and DMSO. Cell Calcium 14:101–110
Nussenzveig DR, Heinflink M, Gershengorn MC (1993) Agonist-stimulated internalization of the thyrotropin-releasing hormone receptor is dependent on two domains in the receptor carboxyl terminus. J Biol Chem 268:2389–2392
Oron Y, Gillo B, Straub RE, Gershengorn MC (1987) Mechanism of membrane electrical response to thyrotropin-releasing hormone in Xenopus oocytes injected with GH3 pituitary cell messenger ribonucleic acid. Mol Endocrinol 1:918–925
Oron Y, Gillo B, Gershengorn MC (1988) Differences in receptor-evoked membrane electrical responses in native and mRNA-injected Xenopus oocytes. Proc Natl Acad Sci USA 85:3820–3824
Palczewski K, Benovic JL (1991) G-protein-coupled receptor kinases. Trends Biochem Sci 16:387–391
Parker I, Yao Y (1994) Relation between intracellular Ca2+ signals and Ca2+ -activated Cl− current in Xenopus oocytes. Cell Calcium 15:276–288
Richardson RM, Kim C, Benovic JL, Hosey MM (1993) Phosphorylation and desensitization of human m2 muscarinic cholinergic receptors by two isoforms of the β-adrenergic receptor kinase. J Biol Chem 268:13650–13656
Roth NS, Campbell PT, Caron MG, Lefkowitz RJ, Lohse MJ (1991) Comparative rates of desensitization of β-adrenergic receptors by the β-adrenergic receptor kinase and the cyclic AMP-dependent protein kinase. Proc Natl Acad Sci USA 8:6201–6204
Ryu SH, Kim UH, Wah MI, Brown AB, Carpenter G, Huang KP, Rhee SG (1990) Feedback regulation of phospholipase C-β by protein kinase C. J Biol Chem 265:17941–17945
Shapira H, Lupu-Meiri M, Gershengorn MC, Oron Y (1990) Activation of two different receptors mobilizes calcium from distinct stores in Xenopus oocytes. Biophys J 57:1281–1285
Shapira H, Lupu-Meiri M, Oron Y (1992) The metabolism of inositol trisphosphate microinjected into Xenopus oocytes. J Basic Clin Physiol Pharmacol 3:119–138
Singer D, Boton R, Moran O, Dascal N (1990) Short and long term desensitization of serotonergic response in Xenopus oocytes injected with brain RNA: roles for inositol 1,4,5-trisphosphate and protein kinase C. Pflügers Arch 416:7–16
Straub RE, Oron Y, Gillo B, Thomson R, Gershengorn MC (1989) Receptor number determines latency and amplitude of the thyrotropin-releasing hormone response in Xenopus oocytes injected with pituitary RNA. Mol Endocrinol 3:907–914
Straub RE, Frech G, Joho RH, Gershengorn MC (1990) Expression cloning of a cDNA encoding the mouse pituitary thyrotropin-releasing hormone receptor. Proc Natl Acad Sci USA 87:9514–9518
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Lipinsky, D., Nussenzveig, D.R., Gershengorn, M.C. et al. Desensitization of the response to thyrotropin-releasing hormone in Xenopus oocytes is an amplified process that precedes calcium mobilization. Pflugers Arch. 429, 419–425 (1995). https://doi.org/10.1007/BF00374158
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00374158