Ma_2011_Biochim.Biophys.Acta_1810_592

BACKGROUND: G protein-coupled receptors fused to a Galpha-subunit are functionally similar to their unfused counterparts. They offer an intriguing view into the nature of the receptor-G protein complex, but their usefulness depends upon the stability of the fusion.
METHODS: Fusion proteins of the M(2) muscarinic receptor and the alpha-subunit of G(i1) were expressed in CHO and Sf9 cells, extracted in digitonin-cholate, and examined for their binding properties and their electrophoretic mobility on western blots.
RESULTS: Receptor fused to native alpha(i1) underwent proteolysis near the point of fusion to release a fragment with the mobility of alpha(i1). The cleavage was prevented by truncation of the alpha-subunit at position 18. Binding of the agonist oxotremorine-M to the stable fusion protein from Sf9 cells was biphasic, and guanylylimidodiphosphate promoted an apparent interconversion of sites from higher to lower affinity. With receptor from CHO cells, the apparent capacity for N-[(3)H]methylscopolamine was 60% of that for [(3)H]quinuclidinylbenzilate; binding at saturating concentrations of the latter was inhibited in a noncompetitive manner at low concentrations of unlabeled N-methylscopolamine.
CONCLUSIONS: A stable fusion protein of the M(2) receptor and truncated alpha(i1) resembles the native receptor-G protein complex with respect to the guanyl nucleotide-sensitive binding of agonists and the noncompetitive binding of antagonists. GENERAL SIGNIFICANCE: Release of the alpha-subunit is likely to occur with other such fusion proteins, rendering the data ambiguous or misleading. The properties of a chemically stable fusion protein support the notion that signaling proceeds via a stable multimeric complex of receptor and G protein.