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

Abstract

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. 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. The homologous desensitization was reversible within 60 s after removal of the agonist. 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. 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]. 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. 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).

Figure 1

Rapid desensitization of the TRH or ACh responses by threshold stimulation of TRH-Rs. Oocytes expressing TRH-Rs alone, or together with m1-Rs, were either challenged with a supramaximal concentration of TRH or ACh alone, or 15 s after a pre-challenge with low [TRH] (0.1–1.0 nM, rapid desensitization – RD). The response to threshold stimulation with low [TRH] alone is shown in the left-most column. The results are the mean±s.e.mean of 22–197 individual measurements in oocytes of 4–13 frogs.

Figure 2

Recovery from rapid desensitization. Oocytes expressing TRH-Rs were pre-challenged with low [TRH] (0.1–1.0 nM). Fifteen seconds later the agonist was washed off and cells were challenged with 10 μM TRH at various times. Desensitization was calculated by comparing the responses to 10 μM TRH after pre-challenge with the response to 10 μM TRH in naive cells. Each point represents results from 12–36 oocytes from 2–5 frogs.

Figure 3

The effect of chelerythrine on rapid homologous desensitization. Oocytes expressing TRH-Rs were challenged either with 10 μM of TRH alone (control) or 15 s following pre-challenge with 0.1–1.0 nM TRH (rapid desensitization, RD). The numbers over the columns represent rapid desensitization as per cent of control responses. Where indicated, oocytes were preincubated for 40 min with chelerythrine (20 μM). Results are presented as mean±s.e.mean of 27–30 oocytes from three frogs.

Figure 4

The effect of chelerythrine on rapid heterologous desensitization. Oocytes expressing TRH-Rs and m1-Rs were challenged either with 10 μM of ACh alone (control) or 15 s following pre-challenge with 0.1–1.0 nM TRH (RD). Where indicated, oocytes were preincubated for 40 min with chelerythrine (20 μM). The numbers over the columns represent desensitization as per cent of control responses. Results are presented as mean±s.e.mean of 29–173 oocytes from 3–13 frogs.

Figure 5

The effect of persistent desensitization on rapid homologous desensitization. Oocytes coexpressing WT and C335Stop TRH-Rs were tested for rapid desensitization (RD) without any treatment (control), following 2 h incubation with 20 μM CDE, or following 40 min incubation with 20 μM chelerythrine. Responses to 10 μM TRH in oocytes expressing WT TRH-Rs alone are shown in the left-most column. Each column represents results obtained in 32–38 oocytes from four frogs.

Figure 6

The effect of persistent desensitization on rapid heterologous desensitization. Oocytes coexpressing m1-Rs and C335Stop TRH-Rs were tested for rapid desensitization (RD) without any treatment (control), following 2 h incubation with 20 μM CDE, or following 40 min incubation with 20 μM chelerythrine. Responses to 10 μM ACh in oocytes expressing m1-Rs alone are shown in the left-most column. Each column represents results obtained in 28–173 oocytes from 3–13 frogs.