In the yeast Saccharomyces cerevisiae, the G protein beta gamma subunits are essential for pheromone signaling. The Galpha subunit Gpa1 can also promote signaling, but the effectors in this pathway are not well characterized. To identify candidate Gpa1 effectors, we expressed the constitutively active Gpa1(Q323L) mutant in each of nearly 5000 gene-deletion strains and measured mating-specific responses. Our analysis reveals a requirement for both the catalytic (Vps34) and regulatory (Vps15) subunits of the sole phosphatidylinositol 3-kinase in yeast. We demonstrate that Gpa1 is present at endosomes, where it interacts directly with both Vps34 and Vps15 and stimulates increased production of phosphatidylinositol 3-phosphate. Notably, Vps15 binds to GDP-bound Gpa1 and is predicted to have a seven-WD repeat structure similar to that of known G protein beta subunits. These findings reveal two new components of the pheromone signaling pathway. More remarkably, these proteins appear to comprise a preformed effector-G beta subunit assembly and function at the endosome rather than at the plasma membrane.

The actions of many extracellular stimuli are elicited by complexes of cell surface receptors, heterotrimeric guanine nucleotide-binding proteins (G proteins), and mitogen-activated protein kinase (MAPK) complexes. Analysis of haploid yeast cells and their response to peptide mating pheromones has produced important advances in the understanding of G protein and MAPK signaling mechanisms. Many of the components, their interrelationships, and their regulators were first identified in yeast. Examples include definitive demonstration of a positive signaling role for G protein betagamma subunits, the discovery of a three-tiered structure of the MAPK module, development of the concept of a kinase-scaffold protein, and the discovery of the first regulator of G protein signaling protein. New and powerful genomic, proteomic, and computational approaches available in yeast are beginning to uncover new pathway components and interactions and have revealed their presence in unexpected locations within the cell. This updated Connections Map in the Database of Cell Signaling includes several major revisions to this prototypical signal response pathway.

The molecular basis of selectivity in G-protein receptor coupling has been explored by comparing the abilities of G-protein heterotrimers containing chimeric G␣ subunits, comprised of various regions of G i1 ␣, G t ␣, and G q ␣, to stabilize the high affinity agonist binding state of serotonin, adenosine, and muscarinic receptors. The data indicate that multiple and distinct determinants of selectivity exist for individual receptors. While the A1 adenosine receptor does not distinguish between G i1 ␣ and G t ␣ sequences, the 5-HT 1A and 5-HT 1B serotonin and M2 muscarinic receptors can couple with G i1 but not G t . It is possible to distinguish domains that eliminate coupling and are defined as "critical," from those that impair coupling and are defined as "important." Domains within the N terminus, ␣4-helix, and ␣4-helix-␣4/␤6-loop of G i1 ␣ are involved in 5-HT and M2 receptor interactions. Chimeric G i1 ␣/G q ␣ subunits verify the critical role of the G␣ C terminus in receptor coupling, however, the individual receptors differ in the C-terminal amino acids required for coupling. Furthermore, the EC 50 for interactions with G i1 differ among the individual receptors. These results suggest that coupling selectivity ultimately involves subtle and cooperative interactions among various domains on both the G-protein and the associated receptor as well as the G-protein concentration.A large number of diverse seven transmembrane-spanning cell surface receptors mediate signaling to a variety of intracellular effectors by coupling to the heterotrimeric guanine nucleotide-binding regulatory proteins (G-proteins) 1 (1). The mechanisms responsible for selectivity in G-protein-mediated signaling pathways are not fully understood (2, 3). Although it is known that at the molecular level the selectivity in G-protein receptor coupling is determined by amino acid sequences of both receptor and G-protein, the individual amino acids involved in this selective recognition have not been completely identified. Different receptor systems and different methodologies indicate that the G␣ subunit C terminus and ␣5-helix (4 -7), N terminus, and ␣N-helix (4, 8 -10), ␣4-helix, and ␣4/ ␤6-loop (11-13), ␣2-helix, and ␣2/␤4-loop (14), ␣3/␤5-loop (15), ␣N/␤1-loop (13) and amino acids 110 -119 from the ␣-helical domain (16) are involved in receptor-coupling selectivity. Some of these domains contact the receptor directly, while others regulate receptor-coupling selectivity indirectly by playing a role in nucleotide exchange. Despite the fact that many of the receptor-interacting domains have been identified, the relationship between receptor subtypes and G␣ domains involved in receptor coupling has not been clearly established. Thus, it is difficult to predict which G␣ domains will be utilized by a specific receptor. Here we propose that individual receptors recognize specific patterns formed by amino acids of G␣ thus making G-protein interface look different for different receptors. The C terminus of G␣ is a well accepted receptor recognit...

Signaling by cell surface receptors and heterotrimeric guanine nucleotide-binding proteins (G proteins) is one of the most exhaustively studied processes in the cell but remains a major focus of molecular pharmacology research. The pheromone-response system in yeast (see the Connections Map at Science's Signal Transduction Knowledge Environment) has provided numerous major advances in our understanding of G protein signaling and regulation. However, the basic features of this prototypical pathway have remained largely unchanged since the mid-1990s. New tools available in yeast are beginning to uncover new pathway components and interactions and have revealed signaling in unexpected locations within the cell.

The molecular basis for selectivity of M1 and M2 muscarinic receptor coupling to heterotrimeric G proteins has been studied using receptors expressed in Sf9 cell membranes and reconstituted with purified chimeric G(alpha) subunits containing different regions of Gi1alpha and Gq(alpha). The abilities of G protein heterotrimers containing chimeric alpha subunits to stabilize the high-affinity state of the receptors for agonist and to undergo receptor stimulated guanine nucleotide exchange was compared with G protein heterotrimers containing either native Gi1alpha or Gq(alpha). The data confirm the importance of the proper context of the C-terminus of Galpha by demonstrating that the C-terminus of Gi1alpha, when placed in the context of Gq(alpha), prevents coupling to muscarinic M1 receptors, while the C-terminus of Gq(alpha), when placed in the context of Gi1alpha, prevents coupling to muscarinic M2 receptors. However, C-terminal amino acids of Gq(alpha) placed in the context of Gi1alpha were not sufficient to allow M1 receptor coupling, nor were C-terminal amino acids of Gi1alpha placed in the context of Gq(alpha) sufficient for M2 receptor coupling. The unique six amino acid N-terminal extension of Gq(alpha) when added to the N-terminus of Gi1alpha neither prevented M2 receptor coupling nor permitted M1 receptor coupling. A Gi1alpha-based chimera containing both N- and C-terminal regions of Gq(alpha) gained the ability to productively couple M1 receptors suggesting that the proper context of both N- and C-termini is required for muscarinic receptor coupling.