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...

The NADPH-dependent respiratory burst oxidase of human neutrophils catalyzes the reduction of oxygen to superoxide using NADPH as the electron donor and is essential for normal host defenses. To gain insight into the function of the various oxidase subunits that are required for the full expression of catalytic activity, we studied the interactions between the 2 Ј ,3 Ј -dialdehyde derivative of NADPH (NADPH dialdehyde) and neutrophil cytosol. NADPH dialdehyde treatment of cytosol resulted in the loss of the ability of the cytosol to participate in cell-free oxidase activation; this inactivation was blocked by NADPH but not by NAD, NADP, or GTP. Partial purification of neutrophil cytosol yielded a single peak which could restore the activity lost in cytosol treated with NADPH dialdehyde. This peak contained p67phox , but not p47 phox or Rac2. Purified recombinant p67 phox was similarly able to restore the activity lost in NADPH dialdehydetreated cytosol and bound [ 32 P]NADPH dialdehyde in a specific fashion. The activity of recombinant p67 phox in cellfree oxidase assays was lost on treatment with NADPH dialdehyde. Together, these data suggest p67 phox contains the catalytic NADPH-binding site of the leukocyte NADPH oxidase. ( J. Clin. Invest. 1996. 98:977-983.)

Subtilisin (Sbt) and Streptomyces subtilisin inhibitor (SSI) were analyzed either alone or together using sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). With all ratios of Sbt to SSI tested, the proteins formed a stoichiometric complex, and migrated abnormally at the top of the gel. Electroblotting and amino acid sequence analysis of the complex band showed both Sbt and SSI present at approximately equal molar ratios. When excess Sbt was present, it migrated as a free but still folded form slightly above the band corresponding to the complex. When excess SSI was present, it migrated as several species with molecular weights smaller than the intact form; in fact, the sequences of some of these species indicated that they lacked different amounts of N-terminal and possibly C-terminal residues.