The G-protein-coupled receptor phosphatase: a protein phosphatase type 2A with a distinct subcellular distribution and substrate specificity.

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 I-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 AB alpha C. PP-2A of this subunit composition has previously been characterized as a soluble enzyme, yet negligible soluble GRP activity was observed. The subcellular distribution and substrate specificity of the GRP suggests significant differences between it and previously characterized forms of PP-2A.

Pleckstrin homology domain-mediated membrane association and activation of the beta-adrenergic receptor kinase requires coordinate interaction with G beta gamma subunits and lipid.

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.

Neutrophil chemotaxis in rhesus macaques (Macaca mulatta).

Chemotactic responses of isolated peripheral blood neutrophils from rhesus macaques (Macaca mulatta) were studied, using a micropore filter method. Cell migration toward zymosan-activated serum was similar to that of human cells, whereas the response to N-formyl methionyl leucyl phenylalanine (FMLP) was weaker than was that in human cells, requiring higher concentrations of FMLP for maximal migration. Optimal FMLP concentrations for attraction of rhesus neutrophils and human neutrophils were 5.0 X 10(-7)M and 1.0 X 10(-8)M, respectively. The chemotactic responses of the 2 neutrophils to complement (zymosan-activated serum) were similar. However, rhesus neutrophils required a higher concentration of the formyl peptide, FMLP, for maximal migratory response.