Effect of the G-Protein, Gαi2, and Gαi3 Subunit Knockdown on Bradykinin-Induced Signal Transduction in Rat-1 Cells

Abstract

The bradykinin (BK) B2 receptor (BKB2R) has been shown to interact with the Gαq subunit family. However, it has remained unclear whether this receptor also interacts with the Gαi subunit family. To further resolve this issue, two antisense expression plasmids were generated. In these, the 5′-untranslated regions of rat Gαi2 and Gαi3 cDNAs were used as specific antisense templates. The plasmids were transfected into Rat-1 cells, which expressed a stably transfected rat BKB2R cDNA and bound BK with a Kd of approximately 3 nM. In these cells, the transfected BKB2R was fully linked to inositol phosphate production, arachidonic acid (ARA) release, and Ca2+ flux. A number of cell lines, each a Gαi2 or Gαi3 knockdown, were isolated. Of these, two cell lines were chosen for study. One, designated 2-E3, displayed over a 70% decrease in the expression of Gαi2 without a change in the expression of Gαi3 or Gαq. Another, 3-G9, exhibited over a 70% decrease of Gαi3 protein without a change in Gαi2 or Gαq expression. Knockdown of either Gαi2 or Gαi3 protein production did not affect the binding of bradykinin. In the Gαi2-depleted 2-E3 cells, BK induced ARA release was reduced by more than 60%. Interestingly, the production of total inositol phosphate in response to BK was also reduced by approximately 35%. However, Gαi2 knockdown had no significant effect on BK-induced Ca2+ influx. In the Gαi3-depleted 3-G9 cells, BK-induced ARA release was decreased by over 50%. In this case [Ca2+]i increase in response to BK was reduced by over 50%. This knockdown also resulted in reduced BK-activated total inositol phosphate production. Moreover, cAMP augmented the BK-induced ARA release. Depletions of Gαi2 and Gαi3 further enhanced this cAMP-dependent BK induction of ARA release. Taken together, these results delineate direct interaction of the BKB2R with both Gαi2 and Gαi3 subunits in addition to the heretofore described interaction of BKB2R with the Gαq subunit family.