Rap1A antagonizes the ability of Ras and Ras-Gap to inhibit muscarinic K+ channels

Abstract

Rap1A is a Ras-related GTP binding protein which has an amino acid sequence identical to that of Ras in the putative "effector" domain (amino acids 32-40). The binding of Rap1A to Ras-GTPase activating protein (GAP) through this domain is a potential mechanism for explaining the observation that Rap1A can antagonize the ability of oncogenic Ras to transform cells. It was recently shown (Yatani, A., Okabe, K., Polakis, P., Halenbeck, R., McCormick, F., and Brown, A. M. (1990) Cell 61, 769-776) that the activation of M2-muscarinic receptor-coupled K+ channels in heart is inhibited by the addition of exogenous Ras and Ras-GAP. We have made use of this system in the present paper to show that Rap1A is able to effectively block this inhibitory action of Ras-GAP. We observed that both Rap1A-GDP and Rap1A-guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) were able to block the inhibitory effect of Ras-GAP upon channel activation. This effect occurred at picomolar concentrations of Rap1A, and the GTP gamma S-bound form of the protein was consistently found to be more potent than the GDP form. A Rap1A Thr35----Ala mutation which bound GTP gamma S did not prevent K+ channel inhibition by Ras-GAP, suggesting that the antagonism by wild type Rap1A involves an interaction with GAP in the effector domain. The effectiveness of Rap1A to inhibit Ras-GAP is dependent upon the amount of Ras-GAP present in the assay and can also be overcome by the addition of GTP-bound N-Ras (GC-43), suggesting a competitive mechanism is operative. Finally, a truncated form of Ras-GAP (GAP32) which is no longer dependent upon Ras for inhibition of the M2-activated K+ channel is also no longer sensitive to blockade by added Rap1A. These data support the concept of GAP as an effector of Ras action and indicate that Rap1A can serve as an inhibitor of Ras action in a system distinct from cell transformation by a competitive mechanism involving the GAP binding domain of Rap1A.