The receptor-mimetic peptide D2N, derived from the cytoplasmic domain of the D(2) dopamine receptor, activates G protein alpha-subunits (G(i) and G(o)) 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 Gbetagamma-subunit: 1) a receptor-prompted movement of Gbetagamma is needed to open up the nucleotide exit pathway ("gear-shift" and "lever-arm" model) or 2) the receptor first engages Gbetagamma and then triggers GDP release by interacting with the carboxyl (C) terminus of Galpha (the "sequential-fit" model). Our results with D2N were compatible with the latter hypothesis. D2N bound to the extreme C terminus of the alpha-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 Mg(2+) control of nucleotide binding. A structural alteration in the outer portion of helix alpha5 (substitution of an isoleucine by proline) blunted the stimulatory action of D2N. This confirms that helix alpha5 links the guanine nucleotide binding pocket to the receptor contact site on the G protein. However, neither the alpha-subunit amino terminus (as a lever-arm) nor Gbetagamma was required for D2N-mediated activation; conversely, assembly of the Galphabetagamma 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 alpha-subunit to destabilize nucleotide binding from the "back side" of the nucleotide binding pocket.