C‐terminal Mutants of Gα12 Selectively Impaired in Ric‐8a Binding

Resistance to inhibitors of cholinesterase-8a (Ric-8a) functions as a non-receptor guanine nucleotide exchange factor for several trimeric G protein α subunits, including those of the Gi, Gq, and G12/13 classes. Ric-8a also has been shown to serve as a chaperone that facilitates synthesis and trafficking of Gα proteins to the plasma membrane. The G12/13 class comprises the α-subunits Gα12 and Gα13, which mediate signaling pathways leading to growth, cytoskeletal changes, and other events. To identify Gα12 determinants of binding to Ric-8a, we immobilized Ric-8a on Sepharose and examined its co-precipitation of Gα12 mutants. Ric-8a showed higher affinity for a constitutively GDP-bound Gα12 than a GTPase-deficient variant, consistent with its role as an activator of nucleotide exchange. Because other investigators found the C-terminus of the Gi α-subunit Gαi1 to mediate its interaction with Ric-8a, we tested C-terminal mutants of Gα12 for loss of Ric-8a binding. Gα12 lacking its 9 C-terminal residues failed to bind Ric-8a, but also was impaired in binding the Gα12 effector protein leukemia-associated RhoGEF (LARG). However, Gα12 mutants harboring cassette substitutions of the sextet Asn-Ala-Ala-Ile-Arg-Ser showed impaired binding to Ric-8a and normal binding to LARG, and Gα12 harboring the C-terminal 9 residues of Gαs showed near-complete loss of Ric-8a binding yet was unimpeded in interaction with LARG and other Gα12 targets. These results show that Gα12 can be mutationally uncoupled from Ric-8a in a selective manner, preserving functional interaction with other Gα12 binding partners. These constructs should facilitate direct studies of the cell biological role of Gα12-Ric-8a interaction. Supported by the GSK Foundation and Lineberger Comprehensive Cancer Center.