Abstract
The stable interaction of a G-protein coupled receptor and a particular partner G-protein was made possible by creating tandems between the α2A adrenergic receptor (α2A-R) and pertussis toxin-resistant mutants of different Gα subunits of heterotrimeric G-proteins. Both α2A-R-Gαoand α2A-R-Gαi proved able to reconstitute agonist-induced voltage-dependent inhibition of N-type calcium channels (CaV2.2) similar to the wild-type α2A-R when expressed in COS-7 cells. The interaction of Gq with the Gi/o signaling pathways was studied by expressing either Gαq or a chimeric construct based on Gαqcontaining the last five amino acids of Gαz, which is activated by α2A-R. It was found that Gαqz5activated by the wild-type α2A-R inhibited CaV2.2 currents in a voltage-independent fashion. Furthermore, Gαqz5 counteracted the voltage-dependent inhibition resulting from α2A-R-Gαoactivation. We subsequently investigated the basis for the behavior of Gαqz5. Our evidence suggests that this occurs as a result of a downstream effect of activation of Gαqz5 because it was blocked by C-terminal construct of phospholipase Cβ1. Furthermore it is likely to occur in part via protein kinase C (PKC) activation, because the PKC activator phorbol dibutyrate mimicked the effects of Gαqz5 in α2A-R-Gαo-transfected cells. Conversely, cells expressing both α2A-R-Gαo and Gαqz5 exhibited a partial restoration of voltage-dependent inhibition in the presence of the PKC inhibitor bisindolylmaleimide I (GF 109203X). The potential sites of phosphorylation are discussed.
Footnotes
- Abbreviations:
- GPCR
- G-protein-coupled receptor
- α2A-R
- α2A-adrenergic receptor
- PTX
- pertussis toxin
- GIRK
- G-protein-coupled inward rectifier K channel
- GFP
- green fluorescent protein
- w.t.
- wild type
- RS-79948-197
- [8aR,12aS,13aS]5,6,8a,9,10,11,12,12a,13,13a-decahydro-12-ethanesulfonyl-3-methoxy-6H-isoquino[2,1-g]-[1,6]naphthyridine hydrochloride)
- GTPγS
- guanosine 5′-O-(3-thio)triphosphate
- HA
- hemagglutinin
- PAGE
- polyacrylamide gel electrophoresis
- PVDF
- polyvinylidene fluoride
- TTBS
- Tween 20/Tris buffered saline
- PDBu
- phorbol dibutyrate
- GF 109203X
- bisindolylmaleimide I
- PKC
- protein kinase C
- PLC-β1ct
- phospholipase C-β1 C terminus
- PP
- prepulse
- Gαt
- Gα-transducin
- PIP2
- phosphatidylinositol 4,5-bisphosphate
- IE
- Ile19Ala, Glu20Ala
- Received September 11, 2002.
- Accepted January 14, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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