| In the yeast, Saccharomyces cerevisiae, there are two RAS genes, RAS1 and RAS2. In S. cerevisiae Ras activates adenylyl cyclase. Adenylyl cyclase is a complex of at least two proteins: the adenylyl cyclase catalytic subunit and an adenylyl cyclase associated protein (CAP) (Field et al., 1990).;Previous studies indicate Cap is a bifunctional molecule. The NH$sb2$ terminus is necessary for full cellular responsiveness to mutant activated RAS proteins. Disruption of the COOH terminus is associated with nutritional and morphological defects. The requirement of Cap in the Ras/adenylyl cyclase pathway and its interaction with an effector of Ras, warranted further study of this molecule.;We cloned a human CAP homologue, designated CAP1 by complementation screening in an S. pombe strain lacking CAP. We demonstrate human CAP1 complements the loss of functions associated with deletion of the COOH terminal CAP domain in S. cerevisiae. When examined for NH$sb2$ terminal function, human CAP1 is incapable of functional complementation of phenotypes associated with deletion of the NH$sb2$ terminus of Cap.;We demonstrate the two hybrid system successfully detects the in vivo association of Cap and adenylyl cyclase. We show the proline rich region of Cap is an SH3 binding domain.;ASP-56, identified by its association with actin, is the pig homologue of human CAP1 (Gieselmann, et al., 1992). We demonstrate by two hybrid and biochemical analysis that yeast Cap and human Cap1 interact with yeast actin.;In a two-hybrid screen designed to detect novel proteins which interact with the NH$sb2$ terminus of human Cap1, and identified a human homologue of talin. In cell extracts prepared from human platelets, we demonstrate that human Cap1 and human talin form an in vivo complex. We show this interaction is conserved through evolution between yeast Cap and a putative yeast, talin homologue, Sla2.;We functionally test the significance of this interaction in S. cerevisiae. We constructed mutants of CAP incapable of interacting with Sla2 and tested them for their ability to complement the loss of endogenous Cap function. Our results suggest that Ras is functionally dependent on Cap and Sla2 to be responsive to an activated $RAS2sp{val19}$ allele. |
