Agonist-dependent regulation of G protein-coupled receptors is dependent on their phosphorylation by G protein-coupled receptor kinases (GRKs). GRK2 and GRK3 are selectively regulated in vitro by free G␤␥ subunits and negatively charged membrane phospholipids through their pleckstrin homology (PH) domains. However, the molecular binding determinants and physiological role for these ligands remain unclear. To address these issues, we generated an array of site-directed mutants within the GRK2 PH domain and characterized their interaction with G␤␥ and phospholipids in vitro. Mutation of several residues in the loop 1 region of the PH domain, including Lys-567, Trp-576, Arg-578, and Arg-579, resulted in a loss of receptor phosphorylation, likely via disruption of phospholipid binding, that was reversed by G␤␥. Alternatively, mutation of residues distal to the C-terminal amphipathic ␣-helix, including Lys-663, Lys-665, Lys-667, and Arg-669, resulted in decreased responsiveness to G␤␥. Interestingly, mutation of Arg-587 in ␤-sheet 3, a region not previously thought to interact with G␤␥, resulted in a specific and profound loss of G␤␥ responsiveness. To further characterize these effects, two mutants (GRK2(K567E/R578E) and GRK2(R587Q)) were expressed in Sf9 cells and purified. Analysis of these mutants revealed that GRK2(K567E/R578E) was refractory to stimulation by negatively charged phospholipids but bound G␤␥ similar to wild-type GRK2. In contrast, GRK2(R587Q) was stimulated by acidic phospholipids but failed to bind G␤␥. In order to examine the role of phospholipid and G␤␥ interaction in cells, wild-type and mutant GRK2s were expressed with a ␤ 2 -adrenergic receptor (␤ 2 AR) mutant that is responsive to GRK2 phosphorylation (␤ 2 AR(Y326A)). In these cells, GRK2(K567E/R578E) and GRK2(R587Q) were largely defective in promoting agonist-dependent phosphorylation and internalization of ␤ 2 AR(Y326A). Similarly, wild-type GRK2 but not GRK2(K567E/R578E) or GRK2(R587Q) promoted morphinedependent phosphorylation of the -opioid receptor in cells. Thus, we have (i) identified several specific GRK2 binding determinants for G␤␥ and phospholipids, and (ii) demonstrated that G␤␥ binding is the limiting step for GRK2-dependent receptor phosphorylation in cells.Diverse extracellular stimuli are perceived at the plasma membrane by G protein-coupled receptors (GPCRs).1 Agonistoccupied receptors promote the activation and dissociation of the heterotrimeric G protein ␣ and ␤␥ subunits, each of which goes on to regulate various effector molecules thereby producing a physiological response. This process is regulated in an agonist-dependent fashion by a family of G protein-coupled receptor kinases (GRKs), which phosphorylate activated GPCRs promoting binding of a second family of proteins, termed arrestins, which serve to uncouple the GPCR from further G protein activation (1, 2). Arrestin binding also promotes receptor internalization, which facilitates the processes of receptor resensitization and down-regulation (1).In general, all G...

Abstract. Ankyrin mediates the attachment of spectrin to transmembrane integral proteins in both erythroid and nonerythroid cells by binding to the (3-subunit of spectrin . Previous studies using enzymatic digestion, 2-nitro-5-thiocyanobenzoic acid cleavage, and rotary shadowing techniques have placed the spectrin-ankyrin binding site in the COOH-terminal third of S-spectrin, but the precise site is not known. We have used a glutathione S-transferase prokaryotic expression system to prepare recombinant erythroid and nonerythroid /3-spectrin from cDNA encoding approximately the carboxy-terminal half of these proteins. Recombinant spectrin competed on an equimolar basis with 125I-labeled native spectrin for binding to erythrocyte membrane vesicles (IM), and also bound ankyrin in vitro as measured by sedimentation velocity experiments. Although full length 0-spectrin could inhibit all spectrin binding to IOVs, recombinant a-spectrin encompassing the complete ankyrin binding domain but lacking the amino-terminal half of the molecule failed to inhibit about 25% of the binding capacity of the IOVs, suggesting that the ankyrinindependent spectrin membrane binding site must lie PECTRIN, the major component ofthe erythrocyte membrane cytoskeleton, is a heterodimer composed of two subunits a, of 280,000 and 246,000 D respectively (Coleman et al., 1989 ; Sahr et al., 1990;Winkelmann et al., 1990). Each subunit is composed predominantly of multiple 106 amino acid residue repeats, c-spectrin containing 22 such repeats and 9-spectrin 17 repeats. Each subunit also contains regions of nonhomologous sequence, and the average repeat-to-repeat sequence identity is -30%. The primary linkage of spectrin to the membrane in erythrocytes is mediated by the binding of0-spectrin with ankyrin . Inherited defects in the linkage of spectrin to ankyrin causes reduced erythrocyte stability and hemolytic disease in both mice (White et al., 1990) and humans Lambert et al., 1990). The recognition that analogues ofboth spectrin

Eotaxin and its receptor, CCR3, are overexpressed in human atherosclerosis, suggesting that eotaxin participates in vascular inflammation. These data demonstrate how genomic differential expression technology can identify novel genes that may participate in the stability of atherosclerotic lesions.

Mechanical overload may change cardiac structure through angiotensin II-dependent and angiotensin IIindependent mechanisms. We investigated the effects of mechanical strain on the gene expression of tenascin-C, a prominent extracellular molecule in actively remodeling tissues, in neonatal rat cardiac myocytes. Mechanical strain induced tenascin-C mRNA (3.9 ؎ 0.5-fold, p < 0.01, n ‫؍‬ 13) and tenascin-C protein in an amplitude-dependent manner but did not induce secreted protein acidic and rich in cysteine nor fibronectin. RNase protection assay demonstrated that mechanical strain induced all three alternatively spliced isoforms of tenascin-C. An angiotensin II receptor type 1 antagonist inhibited mechanical induction of brain natriuretic peptide but not tenascin-C. Antioxidants such as N-acetyl-L-cysteine, catalase, and 1,2-dihydroxy-benzene-3,5-disulfonate significantly inhibited induction of tenascin-C. Truncated tenascin-C promoter-reporter assays using dominant negative mutants of IB␣ and IB kinase ␤ and electrophoretic mobility shift assays indicated that mechanical strain increases tenascin-C gene transcription by activating nuclear factor-B through reactive oxygen species. Our findings demonstrate that mechanical strain induces tenascin-C in cardiac myocytes through a nuclear factor-B-dependent and angiotensin II-independent mechanism. These data also suggest that reactive oxygen species may participate in mechanically induced left ventricular remodeling.Cardiac hypertrophy is an independent risk factor of cardiac morbidity and mortality (1) and is characterized by an increase in myocyte mass and volume, as well as an increase of extracellular matrix proteins such as collagen (2). Angiotensin II is a potent stimulator of cardiac hypertrophy (3), and angiotensin-converting enzyme inhibitors prevent left ventricular hypertrophy in hypertensive animals and humans. For example, Kojima et al. (4) reported that treatment with TCV-116, an angiotensin II receptor type 1 (AT 1 ) 1 antagonist, decreased left ventricular weight, left ventricular wall thickness, and the transverse diameter of cardiac myocytes in spontaneously hypertensive rats.Recent studies (5, 6) indicate that angiotensin II-independent mechanisms may also mediate cardiac hypertrophy. Harada et al. (5) demonstrated that acute pressure overload could induce hypertrophic responses such as induction of c-fos, c-jun, and brain natriuretic peptide (BNP) gene expression, mitogen-activated protein (MAP) kinase activation, and increased heart weight/body weight, in the hearts of AT 1A knockout mice. Harada et al. (6) also reported that there were no significant differences between wild-type mice and AT 1A knockout mice in expression levels of fetal-type cardiac genes, in left ventricular wall thickness and systolic function, or in histological changes such as myocyte hypertrophy and fibrosis.