Abstract
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.
Highlights
Ʈ To whom correspondence should be addressed: Dept. of Medicine, Cardiovascular Division, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115
In order to test the hypothesis that residues in the  subunit that are important for interaction with ␣ may overlap with residues important for activation of effectors, we have analyzed the consequences of mutating residues on the surface of  that interacts with the switch II region of ␣ and residues on the sides of blade 1 of  that contact the amino terminus of the ␣ subunit
Consistent with results in COS-7 cells, H1[K89A]␥2 and H1[L117A]␥2 were approximately equal to wild-type ␥ in activating phospholipase C 2 (PLC2), but the other three mutations on the top surface of 1 were severely blunted in their ability to activate PLC2
Summary
Cell Culture and Transfection—COS-7 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. The medium containing LipofectAMINE was removed, and cells were washed twice with Dulbecco’s modified Eagle’s medium/5% fetal bovine serum. To construct a hexahistidine-tagged 1 subunit (H1), the initial methionine was mutated into glutamine, and at the same time, a HindIII site and a PstI site were introduced. For expression in COS-7 cells, the EcoRV-KpnI fragment of H1 was transferred to the pCDNA3 (Invitrogen) 1 vector using the blunted HindIII and the KpnI sites. The mutated part of the 1 subunit was transferred into pCDNA3 containing H61, using the HindIII and KpnI sites, or KpnI and BamHI as appropriate. Forty to 48 h after transfection, cells were starved for 1 h in methionine- and cysteine-deficient RPMI 1640 medium containing 5% dialyzed fetal bovine serum for 30 min and labeled with 100 Ci/ml Trans[35S]-Label (ICN) for 3 h.
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