Expression of GTPase-deficient Gi2 a subunit (ai2) mutant polypeptides and overexpression of the wild-type oi2 polypeptide in Rat la, Swiss 3T3, and NIH 3T3 fibroblasts altered normal growth regulation and induced a loss of contact inhibition. In Rat la cells (but not in NIH 3T3 or Swiss 3T3 cells), expression of the GTPase-deficient ai2 mutant polypeptides allowed colony formation in soft agar, which correlated with a loss in anchorage dependence and a decreased serum requirement. The altered growth regulatory properties of Rat la cells induced by expression of aj2 mutant polypeptides was not significantly inhibited by cotransfection with a dominant negative Ha-ras mutant polypeptide (Asn-17rasH), indicating that the activated Gi2 membrane signal transduction protein is uniquely capable of altering the regulation of Rat la cell growth by a predominantly c-ras-independent mechanism. The results show that GTPase-deficient ai2 mutant polypeptides have the properties of an oncogene that can induce the phenotypic characteristics of transformation in Rat la cells but that only a subset of these changes is observed with NIH 3T3 and Swiss 3T3 cells.The oncogenic potential of mutant G protein ot subunit polypeptides has been implied from their presence in several neuroendocrine tumors (19,23). Mutations that inhibit the GTPase turn-off function of as, the a chain polypeptide which stimulates adenylyl cyclase, have been identified to occur with fairly high frequency in both pituitary adenomas and thyroid tumors. The role of activated as mutants in the transformation of somatotrophs and thyrocytes is somewhat predictable, because cyclic AMP (cAMP) induces a proliferative response in both cell types (7). Lyons and coworkers also found GTPase-inhibiting mutations in the Gi2 at subunit polypeptide, aOi2, in a subset of ovarian and adrenal cortical tumors (23). It is less apparent how constitutively active, GTPase-deficient ai2 mutant polypeptides could be involved in the transformation of these cell types, since no second messenger system previously characterized to be regulated by Gi2 (adenylyl cyclase inhibition, K+ channels, and PLA2) has been shown to be mitogenic in ovarian or adrenal cortical cells. However, Gi2-like proteins have been implicated in the thrombin-, bombesin-, and lysophosphatidic acid-stimulated mitogenic responses of specific cell types, including Swiss 3T3, NIH 3T3, and Rat la fibroblasts (5,21,37,42 stitutively active ai2 mutant polypeptides would be predicted to impart altered growth control when expressed in cells responding mitogenically to growth factors such as thrombin and lysophosphatidic acid. We have placed mutations that inhibit its intrinsic GTPase activity in the "i2 polypeptide, these are mutations the same as or similar to those present in the as and a2 genes found in a subgroup of neuroendocrine tumors (22, 23). When expressed in three different fibroblast cell lines (Rat la, NIH 3T3, and Swiss 3T3), the GTPasedeficient a(2 mutant polypeptides were capable of altering the growth cha...

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Genetic and structural analysis of the alpha chain polypeptides of heterotrimeric G proteins defines functional domains for GTP/GDP binding, GTPase activity, effector activation, receptor contact and beta gamma subunit complex regulation. The conservation in sequence comprising the GDP/GTP binding and GTPase domains among G protein alpha subunits readily allows common mutations to be made for the design of mutant polypeptides that function as constitutive active or dominant negative alpha chains when expressed in different cell types. Organization of the effector activation, receptor and beta gamma contact domains is similar in the primary sequence of the different alpha subunit polypeptides relative to the GTP/GDP binding domain sequences. Mutation within common motifs of the different G protein alpha chain polypeptides have similar functional consequences. Thus, what has been learned with the Gs and Gi proteins and the regulation of adenylyl cyclase can be directly applied to the analysis of newly identified G proteins and their coupling to receptors and regulation of putative effector enzymes.

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