G-proteins of the q family have been implicated as mediators of bombesin receptors action. We cloned Xenopus G, and G,,, and specifically disrupted the synthesis of either protein with selective antisense oligonucleotides. CC14 antisense inhibited responses mediated by neuromedin B receptor (NMB-R) by 74%, though not by gastrin-releasing peptide receptor (GRP-R). G,,, antisense had little effect on either GRP-R-or NMB-R-mediated responses. This suggests that NMB-R couples to G,_, and that GRP-R and NMB-R show distinct G-protein coupling preferences in the Xenopus oocyte.

Consecutive challenges with thyrotropin-releasing hormone (TRH) of oocytes expressing the TRH receptor (TRH-R) resulted in a pronounced desensitization, manifested as a decrease in chloride current amplitude and an increase in response latency. Exposure to low concentrations of TRH resulted in a marked decrease in the amplitude of the subsequent response to a higher concentration of the agonist, even though the second challenge was given before the onset of the response to the first challenge (within 3 - 15 s). Cellular calcium concentration ([Ca]i) did not increase within this interval, suggesting that calcium was not involved in the desensitization process. The latency of the second response, however, was either unchanged or shortened, implying additive effects of processes initiated by the first challenge. A longer interval (30 s) between the two challenges brought about a more pronounced decrease in amplitude and a prolongation of response latency. The calcium mobilization initiated by a second challenge with a high concentration of the agonist exhibited a longer latency, a lower rate of [Ca]i increase and a lower amplitude. Stimulation of co-expressed cholinergic-muscarinic ml receptors with a low concentration of acetylcholine resulted in a pronounced desensitization of the TRH response (heterologous desensitization). Activation of protein kinase C by beta-phorbol 12-myristate, 13-acetate resulted in a dose-dependent inhibition of the response to TRH, suggesting that protein kinase C was involved in desensitization. Chelerythrine, a specific inhibitor of protein kinase C, abolished a large part of the desensitization. A mutant of the TRH-R that lacks protein kinase C consensus phosphorylation sites in the C-terminal region, exhibited desensitization.(ABSTRACT TRUNCATED AT 250 WORDS)

To dissect the cellular events responsible for the prolonged latency of the response to thyrotropin-releasing hormone (TRH) in Xenopus oocytes we interfered with different steps of the signal transduction pathway. Preincubation of oocytes with cis-vaccenic acid (a membrane-fluidizing agent) shortened the latency, suggesting a contribution of membranal processes. TRH-induced depletion of cellular calcium stores prolonged latency (up to threefold), which returned to control levels upon repletion of the stores. Injection of D-2,3-diphosphoglycerate (PGA), which inhibits inositol (1,4,5)-trisphosphate (InsP3) dephosphorylation, alone evoked a small, prolonged depolarizing current and significantly shortened the latency of the response to TRH. Injection of guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which inactivates guanine nucleotide-binding regulatory proteins, decreased the amplitude of the response and increased latency. Injection of guanosine 5-O-(3-thiotriphosphate) (GTP gamma S) immediately before the challenge with TRH did not shorten the latency of the response. Decreasing the effective receptor density with chlordiazepoxide, an antagonist of the TRH receptor, resulted in an extension of latency, whereas the expression of a large number of TRH receptors by injection of RNA transcribed from cloned receptor DNA (10-100 ng/oocyte) shortened the latency to below 2 s. Our results suggest that the latency of the response to TRH reflects the activation of a late step in the signal transduction sequence, most likely the release of calcium by InsP3. We propose that this process is kinetically controlled by an early rate-limiting event, involving the activation of a guanine nucleotide-binding protein by the TRH receptor.

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Xenopus ooeytes that express mouse thyrotropin-releusing hormone receptors (TRH-Rs) after injection if RNA transcribed from TRH-R eDNA respol~d to THR by a depolarizing current, This r,~sponse is transduced by activation of phosphoinositide-specifie phospholipase C and utilizes an as yet unidentified endogenous guanine nucleotidc-binding regulatory (G) protein(s), The g subunit of G;t and Gq have recently been shown to couple receptors to activation of phospholipase C. To determine whether there are functional differen~ between thg~¢ proteins, we have co-expressed the TRH-R with either rr~ or ct~. =tt potentiated the response to TRH {by 61+16%), while ~ inhibited the n.-spons¢ (by 37±9%). The changes in amplitudes were accompanied by inverse changes in response laten¢ies, These data show that ~t and ~zq differentially modulate signal transduction in Xenopus oocytes.