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  • Title: Effect of the G-protein, G alpha(i2), and G alpha(i3) subunit knockdown on bradykinin-induced signal transduction in rat-1 cells.
    Author: Yang X, Taylor L, Polgar P.
    Journal: Mol Cell Biol Res Commun; 1999 Jun; 1(3):227-36. PubMed ID: 10425231.
    Abstract:
    The bradykinin (BK) B2 receptor (BKB2R) has been shown to interact with the G alpha(q) subunit family. However, it has remained unclear whether this receptor also interacts with the G alpha(i) subunit family. To further resolve this issue, two antisense expression plasmids were generated. In these, the 5'-untranslated regions of rat G alpha(i2) and G alpha(i3) 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 G alpha(i2) or G alpha(i3) 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 G alpha(i2) without a change in the expression of G alpha(i3) or G alpha(q). Another, 3-G9, exhibited over a 70% decrease of G alpha(i3) protein without a change in G alpha(i2) or G alpha(q) expression. Knockdown of either G alpha(i2) or G alpha(i3) protein production did not affect the binding of bradykinin. In the G alpha(i2)-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, G alpha(i2) knockdown had no significant effect on BK-induced Ca2+ influx. In the G alpha(i3)-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 G alpha(i2) and G alpha(i3) further enhanced this cAMP-dependent BK induction of ARA release. Taken together, these results delineate direct interaction of the BKB2R with both G alpha(i2) and G alpha(i3) subunits in addition to the heretofore described interaction of BKB2R with the G alpha(q) subunit family.
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