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

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Abstract
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. 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. CDE abolished heterologous desensitization induced by C335Stop TRH‐R on muscarinic m1‐receptor (m1‐R) co‐expressed in Xenopus oocytes. 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. 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. 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. Western analysis indicated that the α subtype of protein kinase C was down‐regulated in cells expressing C335Stop TRH‐Rs. Following a 5 min exposure to PMA, the residual αPKC translocated to the particular fraction. 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. British Journal of Pharmacology (1999) 126, 1097–1106; doi:10.1038/sj.bjp.0702415

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