Li Y, Sternweis P M, Charnecki S, Smith T F, Gilman A G, Neer E J, Kozasa T
Department of Medicine, Harvard Medical School and Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
J Biol Chem. 1998 Jun 26;273(26):16265-72. doi: 10.1074/jbc.273.26.16265.
Heterotrimeric G proteins, composed of alpha and betagamma subunits, forward signals from transmembrane receptors to intracellular effector enzymes and ion channels. Free betagamma activates downstream targets, but its action is terminated by association with GDP-liganded alpha subunits. Because alpha can inhibit activation of many effectors by betagamma, it is likely that the alpha subunit binding surfaces on betagamma overlap the surfaces necessary for effector activation. To test this hypothesis, we mutated residues on beta shown to contact alpha in the recently published crystal structures of the alphabetagamma heterotrimer (Wall, M. A., Coleman, D. E., Lee, E., Iniguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell 83, 1047-1058; Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319.). The alpha subunit binds to the flat, top surface of the toroidal beta subunit and also extends a helix along the side of the beta subunit at blade 1. We mutated four residues on the top surface of beta (Hbeta1[L117A], Hbeta1[D228R], Hbeta1[D246S], and Hbeta1[W332A]) and two residues on the side of beta that contacts alpha (Hbeta1[N88A/K89A]). Each of the mutant proteins was able to form beta gamma dimers, but they differed in their ability to bind alpha and to activate phospholipase C beta2 (PLCbeta2), PLCbeta3, and adenylyl cyclase II. Mutation of residues along the side of the torus at blade 1 diminish affinity for alpha but do not prevent activation of any of the effectors. Mutations on the alpha binding surface differentially affected PLCbeta2, PLCbeta3, and adenylyl cyclase II. Residues that affect PLCbeta and adenylyl cyclase II activity are found on opposite sides of the central tunnel, suggesting that PLC and adenylyl cyclase, like the alpha subunit, make many contacts on the top surface. None of the mutations affected the ability of betagamma to inhibit adenylyl cyclase I. We conclude that alpha, PLCbeta2, PLCbeta3, and adenylyl cyclase II share an interaction on the top surface of beta. The importance of individual residues is different for alpha binding and for effector activation and differs even between closely related isoforms of the same effector.
异源三聚体G蛋白由α和βγ亚基组成，可将跨膜受体的信号传递给细胞内效应酶和离子通道。游离的βγ激活下游靶点，但其作用会因与结合GDP的α亚基结合而终止。由于α可抑制βγ对许多效应器的激活，因此βγ上与α亚基结合的表面可能与效应器激活所需的表面重叠。为了验证这一假设，我们对β亚基上在最近发表的αβγ异源三聚体晶体结构中显示与α接触的残基进行了突变（沃尔，M.A.，科尔曼，D.E.，李，E.，伊尼格斯-卢希，J.A.，波斯纳，B.A.，吉尔曼，A.G.，和斯普朗，S.R.（1995年）《细胞》83卷，1047 - 1058页；兰布赖特，D.G.，桑德克，J.，博姆，A.，斯基巴，N.P.，哈姆，H.E.，和西格勒，P.B.（1996年）《自然》379卷，311 - 319页）。α亚基结合到环形β亚基的平坦顶面，并沿着β亚基叶片1的侧面延伸一条螺旋。我们对β亚基顶面上的四个残基（Hβ1[L117A]、Hβ1[D228R]、Hβ1[D246S]和Hβ1[W332A]）以及β亚基侧面与α接触的两个残基（Hβ1[N88A/K89A]）进行了突变。每个突变蛋白都能够形成βγ二聚体，但它们在结合α以及激活磷脂酶Cβ2（PLCβ2）、PLCβ3和腺苷酸环化酶II的能力上有所不同。叶片1处环形侧面的残基突变会降低对α的亲和力，但不会阻止任何效应器的激活。α结合表面上的突变对PLCβ2、PLCβ3和腺苷酸环化酶II有不同的影响。影响PLCβ和腺苷酸环化酶II活性的残基位于中央通道的相对两侧，这表明PLC和腺苷酸环化酶与α亚基一样，在顶面上有许多接触点。没有一个突变影响βγ抑制腺苷酸环化酶I的能力。我们得出结论，α、PLCβ2、PLCβ3和腺苷酸环化酶II在β亚基的顶面上存在共同的相互作用。对于α结合和效应器激活，各个残基的重要性不同，甚至在同一效应器的密切相关同工型之间也有所不同。
