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

Institute of Pharmacology, Center of Biomolecular Medicine and Pharmacology, Medical University of Vienna (C.N., R.K., Q.Y., E.F., M.F.); and Boehringer-Ingelheim Austria GmbH, Vienna, Austria (H.A.)

The receptor-mimetic peptide D2N, derived from the cytoplasmic domain of the D₂ 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 Mg²⁺ 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.

Address correspondence to: Dr. C. Nanoff, Medical University of Vienna, Institute of Pharmacology, Waehringer Strae 13, A-1090 Vienna, Austria. E-mail: christian.nanoff{at}meduniwien.ac.at

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