Response to lysophosphatidic acid in Xenopus oocytes and its rapid desensitization: The role of Gq and Go G‐protein families

Abstract

Native Xenopus oocytes exhibit dose-dependent depolarizing current responses to lysophosphatidic acid (LPA), with EC50 = 0.18 microM. Responses to LPA were subject to pronounced rapid desensitization. When oocytes were challenged with 5 nM LPA, the response was <10% of the maximal. Subsequent addition of 0.5 microM LPA resulted in 50-70% desensitization, when compared to naïve controls. Injection of antisense oligodeoxyoligonucleotides (ASODNs) targeted at either of the two endogenous LPA receptors inhibited the LPA response by approximately 50%, but did not alter the degree of rapid desensitization. To study the involvement of G-proteins in rapid homologous desensitization of responses to LPA, we selectively depleted native G-proteins by injection of specific ASDONs. Injection of ASDONs targeted at Galphaq family mRNAs (mainly Galpha11) reduced the response to 0.5 microM LPA by 50%. ASDONs targeted at either Galphao or Galphao1 caused a large decrease in the amount of their cognate mRNAs and the Galphao family proteins, while the response to LPA was inhibited by up to 30%. Injection of ASDONs targeted at Galphao1 mRNA decreased rapid desensitization from 69 to 23%, while pertussis toxin (PTX) completely abolished it. Expression of two dominant negative mutants of the human Galphao family homologs either decreased or virtually abolished rapid desensitization. Microinjection of CaCl(2) demonstrated that 50% of rapid desensitization could be attributed to inhibition of Ca(2+) activation of chloride channels. We propose that the apparent degenerate coupling of different G-proteins to LPA receptors in Xenopus oocytes actually serves both the generation of the response (by Gq and Go G-protein families) and its desensitization (mostly by Go G-protein family).

Go G‐proteins mediate rapid heterologous desensitization of G‐protein coupled receptors in Xenopus oocytes

“…ASODNs sequences (3 0 -atgctgtgtgtgcgctacg-5 0 for Gao; 3 0 -tgcttggatataagcagca-5 0 for Gao1), RT-PCR, quantitative realtime PCR, Western analysis, and preparation of vectors expressing human dominant negative mutation of Go proteins were published previously (Itzhaki Van-Ham et al, 2004a). The sequences of all other ASODNs were previously published (Shapira et al, 1998;Itzhaki Van-Ham et al, 2004a).…”

“…The sequences of all other ASODNs were previously published (Shapira et al, 1998;Itzhaki Van-Ham et al, 2004a).…”

“…We have previously studied rapid homologous desensitization of native LPA-Rs or expressed TRH-Rs (Lipinsky et al, 1995;Itzhaki Van-Ham et al, 2004a). We also described heterologous rapid desensitization of threshold concentrations of ACh or TRH on subsequent responses to high TRH or ACh, respectively (Lipinsky et al, 1995).…”

“…The threshold concentrations of agonists were chosen to minimally activate calcium mobilization pathway and to produce chloride current responses in oocytes that were 10% or less (typically 5%) of the control responses to optimal agonist concentrations (not shown, also see Itzhaki Van-Ham et al, 2004a,b for response to LPA). Our protocol for rapid desensitization was shown to produce a delayed and blunted cellular calcium transient, with markedly slower onset kinetics (Lipinsky et al, 1995).…”

“…As rapid homologous desensitization of the native response to LPA is mediated by the Go family of Gproteins (Itzhaki Van-Ham et al, 2004a), we investigated the role of Go subtypes in rapid heterologous desensitization in Xenopus oocytes. The active protomer of pertussis toxin (PTX) was used to study the involvement of the Gi/o families and ASODNs targeted at either Xenopus Gao or Gao1 transcripts (Itzhaki Van-Ham et al, 2004a) were used to directly probe the involvement of G-proteins of the Go family.…”