The receptor-mimetic peptide D2N, derived from the cytoplasmic domain of the D 2 dopamine receptor, activates G protein ␣-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 G␤␥-subunit: 1) a receptor-prompted movement of G␤␥ is needed to open up the nucleotide exit pathway ("gear-shift" and "lever-arm" model) or 2) the receptor first engages G␤␥ and then triggers GDP release by interacting with the carboxyl (C) terminus of G␣ (the "sequential-fit" model). Our results with D2N were compatible with the latter hypothesis. D2N bound to the extreme C terminus of the ␣-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 2ϩ control of nucleotide binding. A structural alteration in the outer portion of helix ␣5 (substitution of an isoleucine by proline) blunted the stimulatory action of D2N. This confirms that helix ␣5 links the guanine nucleotide binding pocket to the receptor contact site on the G protein. However, neither the ␣-subunit amino terminus (as a lever-arm) nor G␤␥ was required for D2N-mediated activation; conversely, assembly of the G␣␤␥ 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 ␣-subunit to destabilize nucleotide binding from the "back side" of the nucleotide binding pocket.Heterotrimeric G proteins transmit signals from cell surface receptors to intracellular effectors. To activate the G protein, the receptor catalyzes the replacement of Mg-GTP for GDP bound to the G protein ␣-subunit. Because the guanine nucleotide binding pocket is too remote to be directly contacted by the cytoplasmic peptide loops of the receptor, the receptor has to act at a distance (Iiri et al., 1998); however, the exact molecular mechanism is not well defined. The "lever-arm" and "gear-shift" models have been inferred from a structural analogy of the G protein heterotrimer to that of monomeric GTP binding proteins in a complex with their guanine nucleotide exchange factors (Rondard et al., 2001;Cherfils and Chabre, 2003); they predict that the activated receptor (R*) releases GDP by using the ␤␥-subunit to directly (Rondard et al., 2001) or indirectly (Cherfils and Chabre, 2003) distort the ␣-subunit switch regions (I and II).An alternative hypothesis suggests that R* may use a discrete "latch" on the surface of the ␣-subunit and thus prompt GDP release from the "back side" of the nucleotide binding pocket (Sprang, 1997). There are several candidate regions, each marked by the receptor "footprint" [the carboxyl (C) terminus, the amino (N)-terminal extension, loops connecting helix ␣4 to strand ␤6 and ␣2 to ␤4, respectively, as well as side chains linking the N terminus to helix ␣1; cf. Conklin et al., ...