The Carboxyl Terminus of the Gα-Subunit Is the Latch for Triggered Activation of Heterotrimeric G Proteins

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Abstract
The receptor-mimetic peptide D2N, derived from the cytoplasmic domain of the D2 dopamine receptor, activates G protein α-subunits (Gi and Go) directly. Using D2N, we tested the current hypotheses on the mechanism of receptor-mediated G protein activation, which differ by the role assigned to the Gβγ-subunit: 1) a receptor-prompted movement of Gβγ is needed to open up the nucleotide exit pathway (“gear-shift” and “lever-arm” model) or 2) the receptor first engages Gβγ and then triggers GDP release by interacting with the carboxyl (C) terminus of Gα (the “sequential-fit” model). Our results with D2N were compatible with the latter hypothesis. D2N bound to the extreme C terminus of the α-subunit and caused a conformational change that was transmitted to the switch regions. Hence, D2N led to a decline in the intrinsic tryptophan fluorescence, increased the guanine nucleotide exchange rate, and modulated the Mg2+ control of nucleotide binding. A structural alteration in the outer portion of helix α5 (substitution of an isoleucine by proline) blunted the stimulatory action of D2N. This confirms that helix α5 links the guanine nucleotide binding pocket to the receptor contact site on the G protein. However, neither the α-subunit amino terminus (as a lever-arm) nor Gβγ was required for D2N-mediated activation; conversely, assembly of the Gαβγ heterotrimer stabilized the GDP-bound species and required an increased D2N concentration for activation. We propose that the receptor can engage the C terminus of the α-subunit to destabilize nucleotide binding from the “back side” of the nucleotide binding pocket.