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 m1-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 alpha subtype of protein kinase C was down-regulated in cells expressing C335Stop TRH-Rs. Following a 5 min exposure to PMA, the residual alphaPKC 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.