The pleckstrin homology (PH) domain is an approximately 100-amino-acid region of sequence homology present in numerous proteins of diverse functions, which forms a discrete structural module. Several ligands capable of binding to PH domain-containing proteins have been identified including phosphatidylinositol 4,5-bisphosphate (PIP2) and the G beta gamma subunits of heterotrimeric G proteins (G beta gamma), which bind to the amino and carboxyl termini of the PH domain, respectively. Here we report that the binding of G beta gamma and lipid to the PH domain of the beta-adrenergic receptor kinase (beta ARK) synergistically enhances agonist-dependent receptor phosphorylation and that both PH domain-binding ligands are required for membrane association of the kinase. PIP2 and to a lesser extent phosphatidylinositol 4-phosphate, phosphatidylinositol, and phosphatidic acid were the only lipids tested capable, in the presence of G beta gamma, of enhancing beta ARK activity. In contrast, the Km and Vmax for phosphorylation of a soluble beta ARK substrate (casein) was not altered in either the presence or absence of G beta gamma and/or PIP2. A fusion protein of the beta ARK containing an intact PH domain inhibits G beta gamma/PIP2-dependent beta ARK activity. In contrast, a mutant fusion protein in which a tryptophan residue, invariant in all PH domain sequences, is mutated to alanine shows no inhibitory activity. The requirement for the simultaneous presence of two PH domain binding ligands represents a previously unappreciated mechanism for effecting membrane localization of a protein and may have relevance to other PH domain-containing proteins.

Prostate cancer is a leading killer of men in the industrialized world. Underlying this disease is the aberrant action of the androgen receptor (AR). AR is distinguished from other nuclear receptors in that after hormone binding, it preferentially responds to a specialized set of coactivators bearing aromatic-rich motifs, while responding poorly to coactivators bearing the leucine-rich “NR box” motifs favored by other nuclear receptors. Under normal conditions, interactions with these AR-specific coactivators through aromatic-rich motifs underlie targeted gene transcription. However, during prostate cancer, abnormal association with such coactivators, as well as with coactivators containing canonical leucine-rich motifs, promotes disease progression. To understand the paradox of this unusual selectivity, we have derived a complete set of peptide motifs that interact with AR using phage display. Binding affinities were measured for a selected set of these peptides and their interactions with AR determined by X-ray crystallography. Structures of AR in complex with FxxLF, LxxLL, FxxLW, WxxLF, WxxVW, FxxFF, and FxxYF motifs reveal a changing surface of the AR coactivator binding interface that permits accommodation of both AR-specific aromatic-rich motifs and canonical leucine-rich motifs. Induced fit provides perfect mating of the motifs representing the known family of AR coactivators and suggests a framework for the design of AR coactivator antagonists.

Phosphorylation of G-protein-coupled receptors plays an important role in regulating their function. In this study the G-protein-coupled receptor phosphatase (GRP) capable of dephosphorylating G-protein-coupled receptor kinase-phosphorylated receptors is described. The GRP activity of bovine brain is a latent oligomeric form of protein phosphatase type 2A (PP-2A) exclusively associated with the particulate fraction. GRP activity is observed only when assayed in the presence of protamine or when phosphatase-containing fractions are subjected to freeze/thaw treatment under reducing conditions. Consistent with its identification as a member of the PP-2A family, the GRP is potently inhibited by okadaic acid but not by 1-2, the specific inhibitor of protein phosphatase type 1. Solubilization of the membrane-associated GRP followed by gel filtration in the absence of detergent yields a 150-kDa peak of latent receptor phosphatase activity. Western blot analysis of this phosphatase reveals a likely subunit composition of ABaC. of this subunit composition has previously been characterized as a soluble enzyme, yet negligible soluble GRP activity was observed. The subcellular distribution and substrate specifi'city of the GRP suggests significant differences between it and previously characterized forms of PP-2A.Exposure of the f32-adrenergic receptor (,BAR) receptor to agonists results in a rapid decline in receptor responsiveness, a process that appears to involve receptor phosphorylation (1, 2). In addition to the second messenger-dependent protein kinases (1), agonist-specific phosphorylation of this receptor can also be effected by the P3AR kinase (PARK), a member of the family of second messenger-independent G-proteincoupled receptor kinases (GRKs).Despite the considerable progress that has been made in identifying and characterizing the mechanism of action of ,BARK, little is known of the phosphatases responsible for reversing this phosphorylation event. Resensitization, presumably due to dephosphorylation of the ,BAR, has been shown to occur rapidly upon removal of agonists (3). Regulation of receptor phosphatase activity, therefore, represents an important potential mechanism for modulating receptor function.In this study, we characterize the f3ARK-phosphorylated P3AR phosphatase present in extracts ofbovine brain. This phosphatase activity, which is also capable of dephosphorylating ,BARKphosphorylated a2C2-adrenergic receptors (a2C2ARs) and rhodopsin kinase (RK)-phosphorylated rhodopsin, is termed the G-protein-coupled receptor phosphatase (GRP). The enzyme, an oligomeric form of protein phosphatase type 2A (PP-2A), is latent and specifically associated with the particulate fraction. MATERIALS AND METHODSPreparation of 32P-Labeled Substrates. Purified reconstituted P3AR (200 nM) (4-6) was phosphorylated (500-,ul reaction volume) with purified PARK, PAR kinase 2 (I3ARK-2), RK (7), or cAMP-dependent protein kinase (PKA) (125 nM) as described (8). Reactions were terminated after incubation at 30°C for 20 m...