Inaki Azpiazu

Selective Role of G Protein γ Subunits in Receptor Interaction

Receptor stimulation of nucleotide exchange in a heterotrimeric G protein (αβγ) is the primary event-modulating signaling by G proteins. The molecular mechanisms at the basis of this event and the role of the G protein subunits, especially the βγ complex, in receptor activation are unclear. In a reconstituted system, a purified muscarinic receptor, M2, activates G protein heterotrimers αi2β1γ5 and αi2β1γ7 with equal efficacy. However, when the α subunit type is substituted with αo, αoβ1γ7 shows a 100% increase in M2-stimulated GTP hydrolysis compared with αoβ1γ5. Using a sensitive assay based on βγ complex stimulation of phospholipase C activity, we show that both β1γ5 and β1γ7 form heterotrimers equally well with αo and αi. These results indicate that the γ subunit interaction with a receptor is critical for modulating nucleotide exchange and is influenced by the subunit-type composition of the heterotrimer.

A G Protein γ Subunit-specific Peptide Inhibits Muscarinic Receptor Signaling

Muscarinic acetylcholine receptors modulate the function of a variety of effectors through heterotrimeric G proteins. A prenylated peptide specific to the G protein γ5 subunit type inhibits G protein activation by the M2 muscarinic receptor in a reconstitution assay. Scrambling the amino acid sequence of the peptide significantly reduces the efficacy of the peptide. The peptide does not disrupt the G protein heterotrimer. In cultured sympathetic neurons, the γ5 peptide inhibits modulation of Ca2+ current by the M4 receptor. Peptide activity is specific, the scrambled peptide and peptides specific to two other members of the G protein γ subunit family are significantly less effective. The γ5 peptide has no effect on Ca2+ current modulation by the α2-adrenergic and somatostatin receptors. In addition, the γ5 peptide inhibits muscarinic receptor signaling in spinal cord slices with specificity. These results support a specific role for G protein γ subunit types in signal transduction, most likely at the receptor-G protein interface.