Differential coupling of the extreme C-terminus of G protein α subunits to the G protein-coupled melatonin receptors

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Melatonin receptors interact with pertussis toxin-sensitive G proteins to inhibit adenylate cyclase. However, the G protein coupling profiles of melatonin receptor subtypes have not been fully characterised and alternative G protein coupling is evident. The five C-terminal residues of Gα subunits confer coupling specificity to G protein-coupled receptors. This report outlines the use of Gαs chimaeras to alter the signal output of human melatonin receptors and investigate their interaction with the C-termini of Gα subunits. The Gαs portion of the chimaeras confers the ability to activate adenylate cyclase leading to cyclic AMP production. Co-transfection of HEK293 cells expressing MT 1 or MT 2 melatonin receptors with Gαs chimaeras and a cyclic AMP activated luciferase construct provided a convenient and sensitive assay system for identification of receptor recognition of Gα C-termini. Luciferase assay sensitivity was compared with measurement of cyclic AMP elevations by radioimmunoassay. Differential interactions of the melatonin receptor subtypes with Gα chimaeras were observed. Temporal and kinetic parameters of cyclic AMP responses measured by cyclic AMP radioimmunoassay varied depending on the Gαs chimaeras coupled. Recognition of the C-terminal five amino acids of the Gα subunit is a requisite for coupling to a receptor, but it is not the sole determinant.