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

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 Ca2+ concentrations ([Ca2+](i)) in AtT20 cells stably transfected with C335Stop TRH-R cDNA, or Ca2+- 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[Ca2+](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 [Ca2+](i) and a decrease in TRH response amplitude. 3 CDE abolished heterologous desensitization induced by C335Stop TRH-R on muscarinic ml-receptor (ml-R) co-expressed in Xenopus oocytes. 4 Chelation of extracellular calcium with EGTA caused a rapid decrease in [Ca2+](i) and a concomitant increase in the response to TRH in AtT20 cells expressing C335Stop TRH-Rs. 5 Chelerythrine, a specific 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 effect 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 oc subtype of protein kinase C was down-regulated in cells expressing C335Stop TRH-Rs. Following a 5 min exposure to PMA, the residual alpha PKC 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 [Ca2+](i) and PKC.