Identification of determinants in the alpha-subunit of Gq required for phospholipase C activation.
A series of chimeras between a constitutively active mutant of the alpha-subunit of Gq and the alpha-subunit of Gs was constructed to identify the domains in alphaq specifically involved in interaction with its effector phosphoinositide 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 alphaq contained the PLC interaction sites, whereas the residues for activation of adenylyl cyclase were in the Ile235-Leu294 sequence of alphas. Alanine scanning mutagenesis of the Ile217-Lys276 region of alphaq 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 alphaq, alphas, and alphat showed that the PLC-interacting residues identified in alphaq are different from the corresponding residues in alphas and alphat that are involved in effector activation. Alignment of the sequences of alphaq and alphat, based on the crystal structure of alphat (Noel, J. P., Hamm, H. E., and Sigler, P. D. (1993) Nature 366, 654-663), indicated that the PLC-activating residues of alphaq are located in alpha-helix 3 and its linker to beta-sheet 4, which are adjacent to a switch region whose conformation changes with activation. It is proposed that the selectivity of alphaq for PLC involves relatively few amino acids, but that the effector may interact with other nonselective sequences in the alpha-subunit.
Venkatakrishnan
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Howard Hughes Medical Institute and the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295, USA.
Exton
J H
JH
D000409:Alanine; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D017931:DNA Primers; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007668:Kidney; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015290:Second Messenger Systems; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014357:Trypsin
D017931:DNA Primers; D007295:Inositol Phosphates; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D010727:Phosphoric Diester Hydrolases; D014357:Trypsin; D019204:GTP-Binding Proteins; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D000409:Alanine