Venkatakrishnan G, Exton J H
Howard Hughes Medical Institute and the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295, USA.
J Biol Chem. 1996 Mar 1;271(9):5066-72. doi: 10.1074/jbc.271.9.5066.
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.
构建了一系列Gqα亚基的组成型活性突变体与Gsα亚基之间的嵌合体，以鉴定αq中与效应器磷酸肌醇磷脂酶C（PLC）相互作用的特定结构域。嵌合蛋白的瞬时表达以及HEK-293细胞中肌醇磷酸和cAMP产生量的测定表明，αq的Ile217-Lys276序列包含PLC相互作用位点，而腺苷酸环化酶激活的残基位于αs的Ile235-Leu294序列中。对αq的Ile217-Lys276区域进行丙氨酸扫描诱变，进一步确定了与PLC相互作用特别需要的两个氨基酸簇（Asp243、Asn244、Glu245和Arg256、Thr257）。比较αq、αs和αt的序列表明，在αq中鉴定出的与PLC相互作用的残基不同于参与效应器激活的αs和αt中的相应残基。基于αt的晶体结构（Noel，J.P.，Hamm，H.E.和Sigler，P.D.（1993）Nature 366，654-663）对αq和αt的序列进行比对，表明αq的PLC激活残基位于α螺旋3及其与β折叠4的连接区，该区域与一个随激活而构象改变的开关区相邻。有人提出，αq对PLC的选择性涉及相对较少的氨基酸，但效应器可能与α亚基中的其他非选择性序列相互作用。
