A mutant alpha subunit of Gi2 induces neoplastic transformation of Rat-1 cells.

Abstract

In a recently discovered class of oncogenes, GTPase-inhibiting mutations constitutively activate a subunits of signal-transducing guanine nucleotide-binding proteins (G proteins). Somatic mutations in a subclass of endocrine tumors are found in the arginine-179 codon ofthe a subunit ofGu2 (aj2), creating the putative gip2 oncogene. We have tested the ability ofgip2 to mediate neoplastic transformation of Rat-l and NIH 3T3 fibroblasts in tissue culture. Expression of a mutant aj2 cDNA encoding cysteine in place of arginine-179 (au2-R179C)caused Rat-1 cells to grow to a higher density in monolayer culture, to lose anchorage dependence, and to form tumors when injected subcutaneously into nude mice. In contrast, expression of aj2-R179C failed to alter growth or tumorigenicity of NIH 3T3 cells. We conclude that gip2 is an oncogene, by the criterion that it induces neoplastic transformation of Rat-1 cells. Failure of gip2 to transform NIH 3T3 cells is in keeping with clinical indications that gip2 is a tissue-selective oncogene.Hormone receptors activate heterotrimeric guanine nucleotide-binding proteins (G proteins) by promoting replacement of GDP by GTP in the guanine nucleotide-binding sites of the G proteins' a subunits. Each GTP-bound a subunit then regulates the activity of its specific downstream effector(s) until an intrinsic GTPase activity converts bound GTP to GDP, turning off the a subunit and returning it to its GDP-bound, inactive state. In the a subunit of Gs (as), point mutations (1-4) or cholera toxin-catalyzed covalent modification (5) inhibit this GTPase turnoff mechanism, thereby inducing constitutive activation of adenylyl cyclase, the effector of Gs.Recently discovered mutations in genes for G protein a subunits have revealed an additional class of human oncogenes. As with many other oncogenes, these a subunit mutations presumably constitutively activate mitogenic signaling pathways that are normally regulated by their corresponding protooncogene products. The gsp oncogene results from somatic point mutations that inhibit the GTPase activity of as, causing constitutive activation of adenylyl cyclase. By mimicking mitogenic effects of trophic hormones that normally stimulate cAMP synthesis via Gs-coupled receptors, the gsp oncogene contributes to the growth oftumors derived from pituitary somatotrophs (2, 6-9) and thyroid cells (7).A second putative G protein oncogene, gip2, results from somatic point mutations in the gene for the a subunit of Gi2 (ai2) (7). Three inferences suggest that these a12 mutations create an oncogene: (i) gip2 mutations are found in a substantial proportion (z30%) of a restricted subclass of human tumors-i.e., those derived from the adrenal cortex or from endocrine cells of the ovary (7). (ii) gip2 mutations were found by a method-allele-specific hybridization to tumor DNA-that can only detect clonally expanded alterations in DNA; clonal expansion is a hallmark of neoplasia. (iii) gip2 mutations substitute cysteine or histidine for the arginine residue at positi...

Regulation of cell proliferation by G proteins

“…These activated mutant forms of Ga i2 are denoted as gip2 oncogenes (for Gi protein-2). Unlike gsp, the overexpression of gip2 resulted in the oncogenic transformation of Rat1a cells (Pace et al, 1991;Gupta et al, 1992a,b,c). Surprisingly, gip2 failed to transform other ®broblast cell lines such as NIH3T3 cells.…”

“…Surprisingly, gip2 failed to transform other ®broblast cell lines such as NIH3T3 cells. Nevertheless, it has been shown that the gip2-transformed Rat1a cells can form colonies on soft agar and the injection of these cells has been shown to form tumors in athymic nude mice (Pace et al, 1991;Gupta et al, 1992a). Consistent with these ®ndings, inactivating gip2 protooncogene function has been shown to inhibit cell growth and tumor formation (Hermouet et al, 1996a,b).…”

Interaction of the protein nucleobindin with Gαi2, as revealed by the yeast two‐hybrid system

“…One of these proteins, G~i3, has been localized not only to the plasma membrane but also to the Golgi apparatus, where it appears to be involved in the secretion of proteins [5]. G~i2, which interacts with adenylyl cyclase, causes neoplastic transformation of some types of cells when mutated to a constitutively active state [6]. G~i2 may also be involved in the regulation of cell differentiation and in transmission of signals from %-adrenergic receptors to the p21 ras-mitogen activated protein kinase pathway [7,8].…”

G protein coupled receptor signaling through the Src and Stat3 pathway: role in proliferation and transformation

“…The mutant form of Gai2, which is thought to be oncogenic, was termed gip2 and was found to be over expressed in a small subset of ovarian tumors (Lyons et al, 1990), however this does not appear to be widespread as other populations of ovarian tumors have failed to show expression of gip2 (Shen et al, 1996). Expression of Gai2 in Rat-1 cells results in increased proliferation and transformation of cells (Pace et al, 1991). Additionally expression of dominant negative Gai2 has been shown to inhibit melanoma cell proliferation (Hermouet et al, 1996).…”

“…Expression of activated Gao, ai2, aq, a12, and a13, has been shown to cause transformation of cells (Jiang et al, 1993;Kalinec et al, 1992;Kroll et al, 1992;Pace et al, 1991;Vara Prasad et al, 1994). Additionally expression of constitutively activated G protein receptors have also been shown to cause transformation of cells (Allen et al, 1991;Arvanitakis et al, 1997;Cesarman et al, 1996) Therefore, understanding the signaling mechanism leading to the transformation of cells by G protein a subunits and G protein coupled receptor pathways will be very important.…”