Inverse agonist abolishes desensitization of a constitutively active mutant of thyrotropin‐releasing hormone receptor: role of cellular calcium and protein kinase C

Abstract

1 C335Stop is a constitutively active mutant of the TRH receptor (TRH-R). To investigate the mechanism of the decreased responsiveness of C335Stop TRH-R, we studied cellular Ca 2+ concentrations ([Ca 2+ ] i ) in AtT20 cells stably transfected with C335Stop TRH-R cDNA, or Ca 2+ -activated chloride currents in Xenopus laevis oocytes expressing this mutant receptor after injection of cRNA. The competitive TRH-R binding antagonist, chlorodiazepoxide (CDE), was used as an inverse agonist to study the contribution of constitutive activity to desensitization. 2 Acute treatment with CDE resulted in a rapid (within minutes) decrease in [Ca 2+ ] i and an increase in the response amplitude to TRH with no measurable change in receptor density. Conversely, removal of chronically administered CDE caused a rapid increase in [Ca 2+ ] i and a decrease in TRH response amplitude. 3 CDE abolished heterologous desensitization induced by C335Stop TRH-R on muscarinic m1-receptor (m1-R) co-expressed in Xenopus oocytes. 4 Chelation of extracellular calcium with EGTA caused a rapid decrease in [Ca 2+ ] i and a concomitant increase in the response to TRH in AtT20 cells expressing C335Stop TRH-Rs. 5 Chelerythrine, a speci®c inhibitor of protein kinase C (PKC), reversed the heterologous desensitization of the response to acetylcholine (ACh). The phosphoserine/phosphothreonine phosphatase inhibitor, okadaic acid, abolished the e ect of chelerythrine. 6 Down-regulation of PKC by chronic exposure to phorbol 12-myristate 13-acetate (PMA) or acute inhibition with chelerythrine caused a partial resensitization of the response to TRH. 7 Western analysis indicated that the a subtype of protein kinase C was down-regulated in cells expressing C335Stop TRH-Rs. Following a 5 min exposure to PMA, the residual aPKC translocated to the particular fraction. 8 We propose that cells expressing the constitutively active mutant TRH-R rapidly desensitize their response, utilizing a mechanism mediated by an increase in [Ca 2+ ] i and PKC.

Constitutive Signaling by Kaposi's Sarcoma-associated Herpesvirus G-protein-coupled Receptor Desensitizes Calcium Mobilization by Other Receptors

“…The same phenomenon was found, albeit to a lower level of stimulation, in oocytes and in HEK 293 EM cells (Fig. 3), mouse pituitary AtT20 cells expressing a constitutively active mutant of TRH-R, C335Stop (14). 2 Our data clearly indicate that expression of KSHV-GPCRs causes pronounced (50 -80%) desensitization of responses mediated by other GPCRs (TRH-Rs, m1-Rs, or GRP receptors) coexpressed in the same cell.…”

“…This desensitization was fully or partially abolished by chlordiazepoxide, an inverse agonist of TRH-R (14). The heterologous desensitization caused by coexpression of KSHV-GPCRs with other receptors may therefore be attributed to the constitutive activity of the viral receptor.…”

“…Further incubation with thapsigargin led to a major decrease in fractional efflux rate, indicating that the depleted pools could not support the high rate of efflux. DISCUSSION We previously showed that a constitutively signaling TRH-R mutant caused homologous (of TRH-R itself) and heterologous (of other coexpressed GPCRs) desensitization in Xenopus oocytes (11,14). This desensitization was partially or fully abol- …”

“…This may have been true in HEK 293 EM cells also, because we found a smaller fraction of the cell population responding to TRH in populations expressing TRH-Rs and KSHV-GPCRs than in populations expressing TRH-Rs. Hence, the degree of heterologous desensitization attributable to KSHV-GPCRs greatly exceeded that caused by a constitutively active mutant TRH-R (14). To correlate the desensitization with the constitutive activity of KSHV-GPCR, we studied both basal and agonistinduced 45 Ca 2ϩ efflux as a reporter of cellular Ca 2ϩ mobilization.…”

“…Calcium Mobilization in Mammalian Cells-Calcium mobilization was assayed in HEK 293 EM cells and AtT20 pituitary cells by ratiometric determination of [Ca 2ϩ ] i using fura-2 as previously described (14,15). HEK 293 EM cells were transiently transfected with TRH receptor cDNA (0.25-1 g/ml) or cotransfected with TRH-R (0.25-1 g/ml) and KSHV-GPCR (1 g/ml) cDNAs using the calcium phosphate * The costs of publication of this article were defrayed in part by the payment of page charges.…”