Identification and Characterization of Transducin Functional Cysteines, Lysines, and Acidic Residues by Group-Specific Labeling and Chemical Cross-Linking

Guanine nucleotide binding proteins or G-proteins function as molecular switches in a diverse set of signaling pathways by coupling seven-helix transmembrane receptors to specific intracellular effectors (Kaziro et al., 1991; Dohlman et al., 1991). G-proteins are heterotrimers composed of a-, s-, and y-subunits. Activation of the appropiate receptor causes a GDP molecule bound to the resting form of a G-protein to be exchanged for GTP. As a consequence, the G-protein dissociates to form the a-subunit complexed to GTP, and the sy-dimer. The GTP-bound conformation of the a-subunit is capable of activating or inhibiting a variety of downstream effectors including enzymes as well as ion channels (Birnbaumer, 1992; Hepler & Gilman, 1992; Simon et al., 1991). The released sy-complex can itself activate or modulate some effectors (Logothetis et al., 1987; Tang et al., 1991; Katz et al., 1992). A GTPase-controlled timing mechanism inherent in all a-subunits and, in some cases, modulated by other proteins (Berstein et al., 1992; Arshaysky & Bownds, 1992), returns the GTP-activated a-subunit to the inactive GDP-bound conformation. The a-subunit complexed to GDP reassociates with the sy-complex and forms again the hetero-trimer in its resting state. Conklin & Bourne (1993) proposed a structural model for a general G-protein a-subunit, on the basis of biochemical, immunologic, and molecular genetic observations. This model provided a blurred but revealing view of the orientation of membrane-bound Ga with regard to Gpy, receptors, and effectors.