Substitution of asparagine for serine at position 17 decreased the affinity of rasH p21 for GTP 20-to 40-fold without significantly affecting its affinity for GDP. Transfection of NIH 3T3 cells with a mammalian expression vector containing the Asn-17 rasH gene and a Neor gene under the control of the same promoter yielded only a small fraction of the expected number of G418-resistant colonies, indicating that expression of p21 inhibited cell proliferation. The inhibitory effect of Asn-17 p21 required its localization to the plasma membrane and was reversed by coexpression of an activated ras gene, indicating that the mutant p21 blocked the endogenous ras function required for NIH 3T3 cell proliferation. NIH 3T3 cells transformed by v-mos and v-raf, but not v-src, were resistant to inhibition by Asn-17 p21, indicating that the requirement for normal ras function can be bypassed by these cytoplasmic oncogenes. The Asn-17 mutant represents a novel reagent for the study of ras function by virtue of its ability to inhibit cellular ras activity in vivo. Since this phenotype is likely associated with the preferential affinity of the mutant protein for GDP, analogous mutations might also yield inhibitors of other proteins whose activities are regulated by guanine nucleotide binding.The mammalian rais gene family contains three members, ras , ras and rasN, which encode similar 21,000-dalton proteins referred to as p21's (for a review, see reference 1). These proteins reside on the inner face of the plasma membrane, bind GTP and GDP with an equally high affinity. and display a weak GTP hydrolysis activity (1). These properties are similar to those of G proteins that transduce signals from a wide variety of cell surface receptors to enzymes which affect the metabolism of second messengers, including adenylate cyclase, cyclic GMP phosphodiesterase, and phospholipases C and A2 and may also directly regulate ion channels (for a review, see reference 21). Based on this analogy, it has been proposed that r-as proteins also act as signal-transducing molecules. The fact that r-as genes activated by point mutations confer upon cultured cells some aspects of an oncogenic phenotype implies that their encoded proteins function in the transduction of signals that regulate cell proliferation. Other pathways are also probably affected, since p21 is expressed in nondividing cells (6. 19) and activated rcas genes can induce neuronal differentiation of PC12 cells (2, 22), concomitant with a cessation of cell division.G proteins characterized to date have a common structural design. They exist as oligomers made up of ox, ,B, and y subunits (21). In the inactive form. the a subunit is bound to GDP as well as to the a and -y subunits (21). An excited membrane receptor activates the G protein by enhancing the rate of exchange of bound GDP for GTP. Go then dissociates from 13 and y and alters the activity of its target enzyme or ion channel. The ot chain then hydrolyzes its bound GTP to GDP, thereby terminating activation (21). Although no 1 a...