Rapid desensitization of the TRH receptor and persistent desensitization of its constitutively active mutant

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 affecting the desensitized responses. Chelerythrine or the phosphatase inhibitor, okadaic acid, had little effect 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 effect 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)

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 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 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 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. 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