Differential coupling of the extreme C-terminus of G protein alpha subunits to the G protein-coupled melatonin receptors.
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 Galpha subunits confer coupling specificity to G protein-coupled receptors. This report outlines the use of Galphas chimaeras to alter the signal output of human melatonin receptors and investigate their interaction with the C-termini of Galpha subunits. The Galphas 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 Galphas chimaeras and a cyclic AMP activated luciferase construct provided a convenient and sensitive assay system for identification of receptor recognition of Galpha C-termini. Luciferase assay sensitivity was compared with measurement of cyclic AMP elevations by radioimmunoassay. Differential interactions of the melatonin receptor subtypes with Galpha chimaeras were observed. Temporal and kinetic parameters of cyclic AMP responses measured by cyclic AMP radioimmunoassay varied depending on the Galphas chimaeras coupled. Recognition of the C-terminal five amino acids of the Galpha subunit is a requisite for coupling to a receptor, but it is not the sole determinant.
Drew
Janice E
JE
Phytochemicals and Genomic Stability, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, Scotland, UK. jed@rri.sari.ac.uk
Barrett
Perry
P
Conway
Shaun
S
Delagrange
Philippe
P
Morgan
Peter J
PJ
D002460:Cell Line; D000242:Cyclic AMP / Q000032:analysis / Q000235:genetics; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008156:Luciferases; D008550:Melatonin / Q000494:pharmacology; D008969:Molecular Sequence Data; D011863:Radioimmunoassay; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D044094:Receptors, Melatonin; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D044094:Receptors, Melatonin; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D008156:Luciferases; D019204:GTP-Binding Proteins; D020962:Heterotrimeric GTP-Binding Proteins; D008550:Melatonin