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.

1 We studied rapid desensitization of the thyrotropin-releasing hormone receptor (TRH-R) or the m1-muscarinic receptor (m1-R) to a short challenge of threshold TRH concentration and persistent desensitization due to constitutive activity of a mutant TRH-R. 2 Xenopus oocytes expressing TRH-Rs and/or m1-Rs were challenged for 15 s with threshold concentrations of TRH ([TRH]) and then immediately with supraoptimal [TRH] or acetylcholine ([ACh]). The threshold challenge caused desensitization of 50 ± 57% of responses to subsequent supraoptimal stimulation with TRH or ACh. 3 The homologous desensitization was reversible within 60 s after removal of the agonist. 4 The protein kinase C (PKC) inhibitor, chelerythrine, inhibited the control responses by 30 ± 40%, without aecting the desensitized responses. Chelerythrine or the phosphatase inhibitor, okadaic acid, had little eect on the kinetics of resensitization, indicating limited involvement of PKC. 5 In oocytes coexpressing wild type TRH-Rs or m1-Rs with a constitutively active TRH-R mutant (C335Stop TRH-R), a persistent desensitization (33 ± 57%) of the responses to TRH or ACh was observed. Additionally, there was a complete loss of the rapid desensitization induced by threshold [TRH]. 6 Chlorodiazepoxide (CDE), a competitive binding antagonist of TRH-Rs and an inverse agonist of C335Stop TRH-Rs, abolished the persistent desensitization induced by C335Stop TRH-Rs and enabled the rapid desensitization, conferring the wild type phenotype on C335Stop TRH-Rs. Chelerythrine had qualitatively the same eect as CDE. 7 In conclusion, unlike the rapid desensitization, the persistent desensitization caused by the constitutively active C335Stop TRH-Rs is largely mediated by PKC. It abrogates, however, the rapid desensitization, suggesting a common mechanistic step(s).