Identification of determinants in the α-subunit of Gq required for phospholipase C activation

Abstract

A series of chimeras between a constitutively active mutant of the α-subunit of Gq and the α-subunit of Gs was constructed to identify the domains in αq specifically involved in interaction with its effector phospho-inositide phospholipase C (PLC). Transient expression of the chimeric proteins and measurement of the production of inositol phosphates and cAMP in HEK-293 cells revealed that the Ile217-Lys276 sequence of αq contained the PLC interaction sites, whereas the residues for activation of adenylyl cyclase were in the Ile235-Leu294 sequence of αs. Alanine scanning mutagenesis of the Ile217-Lys276 region of a further identified two clusters of amino acids (Asp243,Asn244,Glu245 and Arg256,Thr257) that were specifically required for interaction with PLC. Comparison of the sequences of αq, αs, and αt showed that the PLC-interacting residues identified in αq are different from the corresponding residues in αs and αt that are involved in effector activation. Alignment of the sequences of αq and αt, based on the crystal structure of αt (Noel, J. P., Hamm, H. E., and Sigler, P. D. (1993) Nature 366, 654-663), indicated that the PLC-activating residues of αq are located in α-helix 3 and its linker to β-sheet 4, which are adjacent to a switch region whose conformation changes with activation. It is proposed that the selectivity of αq for PLC involves relatively few amino acids, but that the effector may interact with other nonselective sequences in the α-subunit.