Mutagenesis of the conserved residue Glu(259) of G(s)alpha demonstrates the importance of interactions between switches 2 and 3 for activation

We previously reported that substitution of Arg(258) within the switch 3 re gion of G(s)alpha impaired activation and increased basal GDP release due t o loss of an interaction between the helical and GTPase domains (Warner, D. R., Weng, G., Yu, S., Matalon, R., and Weinstein, L. S. (1998) J Biol. Che m. 273, 23976-23983). The adjacent residue (Glu(259)) is strictly conserved in G protein alpha-subunits and is predicted to be important in activation . To determine the importance of Glu(259), this residue was mutated to Ala (G(s)alpha-E259A), Gln (G(s)alpha-E259Q), Asp (G(s)alpha-E259D), or Val (G( s)alpha-E259V), and the properties of in vitro translation products were ex amined. The G(s)alpha-E259V was studied because this mutation was identifie d in a patient with Albright hereditary osteodystrophy. 549 eye reconstitut ion assays demonstrated that G(s)alpha-E259D stimulated adenylyl cyclase no rmally in the presence of GTP gamma S but was less efficient with isoproter enol or AlF4-. The other mutants had more severely impaired effector activa tion, particularly in response to AlF4-. In trypsin protection assays, GTP gamma S was a more effective activator than AlF4- for all mutants, with G(s )alpha-E259D being the least severely impaired. For G(s)alpha-E259D, the Al F4--induced activation defect was more pronounced at low Mg2+ concentration s. G(s)alpha-E259D and G(s)alpha-E259A purified from Escherichia coil had n ormal rates of GDP release (as assessed by the rate GrT gamma S binding). H owever, for both mutants, the ability of AlF4- to decrease the rate of GTP gamma S binding was impaired, suggesting that they bound AlF4- more poorly. GrTP gamma S bound to purified G(s)alpha-E259D irreversibly in the presenc e of 1 mM free Mg2+, but dissociated readily at micromolar concentrations. Sucrose density gradient analysis of in, vitro translates demonstrated that all mutants except G(s)alpha-E259V bind to beta gamma at 0 degrees C and w ere stable at higher temperatures. In the active conformation Glu(259) inte racts with conserved residues in the switch 2 region that are important in maintaining both the active state and AlF4- in the guanine nucleotide bindi ng pocket. Although both G(s)alpha Arg(258) and Glu(259) are critical for a ctivation, the mechanisms by which these residues affect G(s)alpha. protein activation are distinct.