A yeast two-hybrid approach was used to discern possible new effectors for the ␥ subunit of heterotrimeric G proteins. Three of the clones isolated are structurally similar to G, each exhibiting the WD40 repeat motif. Two of these proteins, the receptor for activated C kinase 1 (RACK1) and the dynein intermediate chain, coimmunoprecipitate with G␥ using an anti-G antibody. The third protein, AAH20044, has no known function; however, sequence analysis indicates that it is a WD40 repeat protein. Further investigation with RACK1 shows that it not only interacts with G 1 ␥ 1 but also unexpectedly with the transducin heterotrimer G␣ t  1 ␥ 1 . G␣ t alone does not interact, but it must contribute to the interaction because the apparent EC 50 value of RACK1 for G␣ t  1 ␥ 1 is 3-fold greater than that for G 1 ␥ 1 (0.1 versus 0.3 M). RACK1 is a scaffold that interacts with several proteins, among which are activated IIPKC and dynamin-1 (1). IIPKC and dynamin-1 compete with G 1 ␥ 1 and G␣ t  1 ␥ 1 for interaction with RACK1. These findings have several implications: 1) that WD40 repeat proteins may interact with each other; 2) that G␥ interacts differently with RACK1 than with its other known effectors; and/or 3) that the G protein-RACK1 complex may constitute a signaling scaffold important for intracellular responses.Heterotrimeric G proteins are a family of proteins that transduce an extracellular signal to an intracellular response via a seven helical transmembrane G protein-coupled receptor (GPCR).1 Upon activation, the receptor facilitates the exchange of GDP for GTP in the G␣ subunit. G␣ is then thought to dissociate from the G␥ heterodimer allowing both complexes to individually activate a number of effectors (2, 3). Free G␥ interacts with a large assortment of effector proteins, including phospholipases (4), adenylyl cyclases (5), ion channels (6), and G protein-coupled receptor kinases (7). There are, however, G protein-coupled receptor responses, such as MAP kinase activation (8 -10), receptor internalization (11, 12), and organelle transport (13-15) that are mediated through the G␥ subunit but that have not been definitively linked to known G␥ effectors.G is the prototypical member of a family of proteins known as WD40 repeat proteins, which seem to function as adaptors and enzyme regulators (16,17). G is the only WD40 repeat protein whose three-dimensional structure is known, and it exhibits a toroidal bladed -propeller structure, with each blade consisting of 4 anti-parallel -strands (18). Because the WD repeat motif is a structural element of the -propeller, all of these proteins are thought to be -propeller proteins with a variable number of blades. Furthermore, G subunits are known to interact with G␥ subunits, proteins containing a G␥-like domain (19), a pleckstrin homology domain (20), a QXXER domain (found in adenylyl cyclases) (21), and a domain contained within phosducin and its relatives (22). In this work we propose that G␥ also interacts with certain other WD40 repeat prote...