The constitutive signaling of KSHV-GPCR is mediated by Gαq and Gαi3 in Xenopus oocytes

Monica Lupu-Meiri, Maya Cheifetz, Irit Yitzhaki & Yoram Oron

AIMS: To study G-protein(s) coupling to Kaposi sarcoma-associated herpesvirus 8 receptor (KSHV-GPCR). METHODS: KSHV-GPCR was expressed in Xenopus oocytes. Constitutive signaling of the viral receptor was assessed by labeled calcium efflux. The effect of its expression on activation of the Gα pathway was assayed by monitoring responses mediated by endogenous adenosine receptors. Endogenous oocyte G-proteins were depleted by selective knock-out of individual mRNA populations, using antisense phosphorothioate deoxyoligonucleotides (S-ASODNs). The specificity and selectivity of S-ASODNs was assessed by RT-PCR and real-time PCR. RESULTS: KSHV-GPCR, expressed alone in oocytes, caused 4.4-fold increase in labeled calcium basal efflux, which was almost fully inhibited by IP10, demonstrating the receptors constitutive signaling. Expression of KSHV-GPCR and application of IP10 had no effect on adenosine-evoked potassium current, a reporter of adenosine receptor-Gα-cAMP pathway activation. Selective knockout of endogenous Gα, Gαo2, Gαi1, Gα11 and Gα14 had no significant effect on the elevated calcium efflux. Knockout of Gαq and Gαi3 abolished increased calcium efflux by 60 and 40%, respectively. Knockout of Gαo1 inhibited the increased efflux by 30%. CONCLUSIONS: KSHV-GPCR is a constitutively signaling receptor, which couples to Gαq, Gαi3 and, possibly, to Gαo1. This study confirms the coupling of KSHV-GPCR to pertussis-toxin sensitive and insensitive G-proteins. However, in oocytes, both G-protein families couple to the activation of phospholipase C, but not to adenylyl cyclase.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Viral Oncogenesis.

http://www.cancerprev.org/Journal/Issues/26/101/996/4339