Abstract
Characteristically, an individual member of the superfamily of G protein-coupled receptors can interact only with a limited number of the many structurally closely related G protein heterotrimers that are expressed within a cell. Interestingly, the N termini of two G protein alpha subunits, Galphaq and Galpha11, differ from those of other alpha subunits in that they display a unique, highly conserved six-amino acid extension. To test the hypothesis that this sequence element is critical for proper receptor recognition, we prepared a Galphaq deletion mutant (-6q) lacking these first six amino acids. The -6q construct (or wild type Galphaq as a control) was coexpressed (in COS-7 cells) with several different Gi/o- or Gs-coupled receptors, and ligand-induced increases in inositol phosphate production were determined as a measure of G protein activation. Whereas these receptors did not efficiently interact with wild type Galphaq, most of them gained the ability to productively couple to -6q. Additional experiments indicated that the observed functional promiscuity of -6q is not due to overexpression (as compared with wild type Galphaq) or to a lack of palmitoylation. We conclude that the N-terminal extension characteristic for Galphaq/11 proteins is critical for constraining the receptor coupling selectivity of these subunits, indicative of a novel mechanism by which the fidelity of receptor-G protein interactions can be regulated.
Highlights
From the Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892 and the ‡Departments of Medicine and Pharmacology, Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, California 94141-9100
Transfected cells were incubated with the appropriate agonist ligands, and the ability of the different receptors to couple to the two G proteins was determined by measuring increases in inositol phosphate production
Consistent with previous findings [33, 34], this observation suggests that the m2 muscarinic and D2 dopamine receptors do not couple to wild type Gaq (WTq) to a significant extent and that the small increase in PI hydrolysis seen after stimulation of these receptors is most likely due to activation of PLCb by G protein bg subunits released upon receptor-mediated activation of endogenous Gi/o proteins [35, 36]
Summary
Both studies showed that Gq/11-coupled receptors such as the NK2 neurokinin [16] and the m1 muscarinic receptor [17] were able to activate 26q in a fashion identical to WTq. other functional properties, such as their affinity for bg subunits and their ability to activate downstream effectors (e.g. phospholipase Cb (PLCb)), were found to be very similar for the two G protein subunits [16, 17]. Whereas none of the receptors (upon incubation with the appropriate agonist ligands) was able to activate WTq to a significant extent, most of the receptors gained the ability to couple to 26q with considerable efficiency (measured biochemical response: stimulation of phosphatidylinositol (PI) hydrolysis).
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