Journal
MOLECULAR MEMBRANE BIOLOGY
Volume 22, Issue 6, Pages 507-517Publisher
TAYLOR & FRANCIS LTD
DOI: 10.1080/09687860500370604
Keywords
G-proteins; G-protein coupled receptor; signalling; receptor; dissociation
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G-protein coupled receptors (GPCRs) form a ternary complex of agonist, receptor and G-proteins during primary signal transduction at the cell membrane. Downstream signalling is thought to be preceded by the process of dissociation of G alpha and G beta gamma subunits, thus exposing new surfaces to interact with downstream effectors. We demonstrate here for the first time, the dissociation of heterotrimeric G-protein subunits (i.e., G alpha and G beta gamma) following agonist-induced GPCR (alpha(2A)-adrenergic receptor; alpha(2A)-AR) activation in a cell-free assay system. alpha(2A)-AR membranes were reconstituted with the G-proteins (+/- hexahistidine-tagged) G alpha(i1) and G beta(1)gamma(2) and functional signalling was determined following activation of the reconstituted receptor: G-protein complex with the potent agonist UK-14304, and [S-35]GTP gamma S. In the presence of Ni2+-coated agarose beads, the activated his-tagged G alpha(i1)his-[S-35]GTPgS complex was captured on the Ni2+-presenting surface. When his-tagged G beta(1 gamma 2) (G beta(1)gamma(2)his) was used with G alpha(i1), the [S-35]GTP gamma S-bound G alpha(i1) was not present on the Ni2+-coated beads, but rather, it was separated from the beta(1)gamma(2)(his)-beads, demonstrating receptor-induced dissociation of G alpha and G beta gamma subunits. Treatment of the reconstituted alpha(2A)-AR membranes containing G beta(1 gamma 2)his: G alpha(i1) with imidazole confirmed the specificity for the Ni2+ :G-protein surface dissociation of G alpha(i1) from G beta(1)gamma(2)his. These data demonstrate for the first time, the complete dissociation of the G-protein subunits and extend observations on the role of G-proteins in the assembly and disassembly of the ternary complex in the primary events of GPCR signalling.
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