The membrane localization of the G protein α_s subunit is not dependent on its tenir sequence or effector domain

The G_s protein subunit (α_s) sequences conserved in non-myristoylated α subunits (TENIR, residues 369-373) or critical for adenylyl cyclase interaction were investigated as possible sites required for membrane localization. Substitutions were created by site-directed mutagenesis in which the TENIR residues were deleted from α_s or added to the soluble, non-myristoylated α_il. After transfection, COS cells were separated by centrifugation into particulate and soluble fractions. Immunoblots showed that these substitutions did not change the localization: α_s ± TENIR in the particulate fraction, non-myristoylated α_il ± TENIR in the soluble fraction. The constitutively active α_i/α_s chimera (CH4A), containing four regions of α_s sufficient for adenylyl cyclase activation, was mutated to prevent myristoylation (GA-CH4A). Immunoblots of transfected COS cell fractions showed CH4A in the particulate and GA-CH4A in the soluble fraction. While these regions did not lead to memebrane localization, the soluble GA-CH4A could activate adenylyl cyclase in the intact cell and after reconstitution with cyc- membranes.