Mutagenesis of the conserved residue Glu259 of Gsα demonstrates the importance of interactions between switches 2 and 3 for activation.

We previously reported that substitution of Arg(258) within the switch 3 region of G(s)alpha impaired activation and increased basal GDP release due to 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. Chem. 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 examined. The G(s)alpha-E259V was studied because this mutation was identified in a patient with Albright hereditary osteodystrophy. 549 eye reconstitution assays demonstrated that G(s)alpha-E259D stimulated adenylyl cyclase normally in the presence of GTP gamma S but was less efficient with isoproterenol or AlF4-. The other mutants had more severely impaired effector activation, 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 AlF4--induced activation defect was more pronounced at low Mg2+ concentrations. G(s)alpha-E259D and G(s)alpha-E259A purified from Escherichia coli had normal rates of GDP release (as assessed by the rate GrT gamma S binding). However, 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 presence 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 were stable at higher temperatures. In the active conformation Glu(259) interacts with conserved residues in the switch 2 region that are important in maintaining both the active state and AlF4- in the guanine nucleotide binding pocket. Although both G(s)alpha Arg(258) and Glu(259) are critical for activation, the mechanisms by which these residues affect G(s)alpha. protein activation are distinct.