Neil J. Freedman

Phosphorylation and Desensitization of the Human -Adrenergic Receptor INVOLVEMENT OF G PROTEIN-COUPLED RECEPTOR KINASES AND cAMP-DEPENDENT PROTEIN KINASE

Persistent stimulation of the β1-adrenergic receptor (β1AR) engenders, within minutes, diminished responsiveness of the β1AR/adenylyl cyclase signal transduction system. This desensitization remains incompletely defined mechanistically, however. We therefore tested the hypothesis that agonist-induced desensitization of the β1AR (like that of the related β2AR) involves phosphorylation of the receptor itself, by cAMP-dependent protein kinase (PKA) and the β-adrenergic receptor kinase (βARK1) or other G protein-coupled receptor kinases (GRKs). Both Chinese hamster fibroblast and 293 cells demonstrate receptor-specific desensitization of the β1AR within 3-5 min. Both cell types also express βARK1 and the associated inhibitory proteins β-arrestin-1 and β-arrestin-2, as assessed by immunoblotting. Agonist-induced β1AR desensitization in 293 cells correlates with a 2 ± 0.3-fold increase in phosphorylation of the β1AR, determined by immunoprecipitation of the β1AR from cells metabolically labeled with Pi. This agonist-induced β1AR phosphorylation derives approximately equally from PKA and GRK activity, as judged by intact cell studies with kinase inhibitors or dominant negative βARK1 (K220R) mutant overexpression. Desensitization, likewise, is reduced by only 50% when PKA is inhibited in the intact cells. Overexpression of rhodopsin kinase, βARK1, βARK2, or GRK5 significantly increases agonist-induced β1AR phosphorylation and concomitantly decreases agonist-stimulated cellular cAMP production (p < 0.05). Furthermore, purified βARK1, βARK2, and GRK5 all demonstrate agonist-dependent phosphorylation of the β1AR. Consistent with a GRK mechanism, receptor-specific desensitization of the β1AR was enhanced by overexpression of β-arrestin-1 and −2 in transfected 293 cells. We conclude that rapid agonist-induced desensitization of the β1AR involves phosphorylation of the receptor by both PKA and at least βARK1 in intact cells. Like the β2AR, the β1AR appears to bind either β-arrestin-1 or β-arrestin-2 and to react with rhodopsin kinase, βARK1, βARK2, and GRK5.

Phosphorylation and Desensitization of Human Endothelin A and B Receptors EVIDENCE FOR G PROTEIN-COUPLED RECEPTOR KINASE SPECIFICITY

Although endothelin-1 can elicit prolonged physiologic responses, accumulating evidence suggests that rapid desensitization affects the primary G protein-coupled receptors mediating these responses, the endothelin A and B receptors (ETA-R and ETB-R). The mechanisms by which this desensitization proceeds remain obscure, however. Because some intracellular domain sequences of the ETA-R and ETB-R differ substantially, we tested the possibility that these receptor subtypes might be differentially regulated by G protein-coupled receptor kinases (GRKs). Homologous, or receptor-specific, desensitization occurred within 4 min both in the ETA-R-expressing A10 cells and in 293 cells transfected with either the human ETA-R or ETB-R. In 293 cells, this desensitization corresponded temporally with agonist-induced phosphorylation of each receptor, assessed by receptor immunoprecipitation from 32Pi-labeled cells. Agonist-induced receptor phosphorylation was not substantially affected by PKC inhibition but was reduced 40% (p < 0.03) by GRK inhibition, effected by a dominant negative GRK2 mutant. Inhibition of agonist-induced phosphorylation abrogated agonist-induced ETA-R desensitization. Overexpression of GRK2, -5, or -6 in 293 cells augmented agonist-induced ET-R phosphorylation ∼2-fold (p < 0.02), but each kinase reduced receptor-promoted phosphoinositide hydrolysis differently. While GRK5 inhibited ET-R signaling by only ∼25%, GRK2 inhibited ET-R signaling by 80% (p < 0.01). Congruent with its superior efficacy in suppressing ET-R signaling, GRK2, but not GRK5, co-immunoprecipitated with the ET-Rs in an agonist-dependent manner. We conclude that both the ETA-R and ETB-R can be regulated indistinguishably by GRK-initiated desensitization. We propose that because of its affinity for ET-Rs demonstrated by co-immunoprecipitation, GRK2 is the most likely of the GRKs to initiate ET-R desensitization.

Nedd4 Mediates Agonist-dependent Ubiquitination, Lysosomal Targeting, and Degradation of the β2-Adrenergic Receptor

Agonist-stimulated β2-adrenergic receptor (β2AR) ubiquitination is a major factor that governs both lysosomal trafficking and degradation of internalized receptors, but the identity of the E3 ubiquitin ligase regulating this process was unknown. Among the various catalytically inactive E3 ubiquitin ligase mutants that we tested, a dominant negative Nedd4 specifically inhibited isoproterenol-induced ubiquitination and degradation of the β2AR in HEK-293 cells. Moreover, siRNA that down-regulates Nedd4 expression inhibited β2AR ubiquitination and lysosomal degradation, whereas siRNA targeting the closely related E3 ligases Nedd4-2 or AIP4 did not. Interestingly, β2AR as well as β-arrestin2, the endocytic and signaling adaptor for the β2AR, interact robustly with Nedd4 upon agonist stimulation. However, β2AR-Nedd4 interaction is ablated when β-arrestin2 expression is knocked down by siRNA transfection, implicating an essential E3 ubiquitin ligase adaptor role for β-arrestin2 in mediating β2AR ubiquitination. Notably, β-arrestin2 interacts with two different E3 ubiquitin ligases, namely, Mdm2 and Nedd4 to regulate distinct steps in β2AR trafficking. Collectively, our findings indicate that the degradative fate of the β2AR in the lysosomal compartments is dependent upon β-arrestin2-mediated recruitment of Nedd4 to the activated receptor and Nedd4-catalyzed ubiquitination.

Anti–β1-adrenergic receptor antibodies and heart failure: causation, not just correlation

Antibodies specific for the beta(1)-adrenergic receptor are found in patients with chronic heart failure of various etiologies. From work presented in this issue of the JCI, we can now infer that these antibodies actually contribute to the pathogenesis of chronic heart failure. This commentary discusses mechanisms by which these antibodies may engender cardiomyopathy.

Neuropeptide Y Gene Polymorphisms Confer Risk of Early-Onset Atherosclerosis

Neuropeptide Y (NPY) is a strong candidate gene for coronary artery disease (CAD). We have previously identified genetic linkage to familial CAD in the genomic region of NPY. We performed follow-up genetic, biostatistical, and functional analysis of NPY in early-onset CAD. In familial CAD (GENECARD, N = 420 families), we found increased microsatellite linkage to chromosome 7p14 (OSA LOD = 4.2, p = 0.004) in 97 earliest age-of-onset families. Tagged NPY SNPs demonstrated linkage to CAD of a 6-SNP block (LOD = 1.58–2.72), family-based association of this block with CAD (p = 0.02), and stronger linkage to CAD in the earliest age-of-onset families. Association of this 6-SNP block with CAD was validated in: (a) 556 non-familial early-onset CAD cases and 256 controls (OR 1.46–1.65, p = 0.01–0.05), showing stronger association in youngest cases (OR 1.84–2.20, p = 0.0004–0.09); and (b) GENECARD probands versus non-familial controls (OR 1.79–2.06, p = 0.003–0.02). A promoter SNP (rs16147) within this 6-SNP block was associated with higher plasma NPY levels (p = 0.04). To assess a causal role of NPY in atherosclerosis, we applied the NPY1-receptor–antagonist BIBP-3226 adventitially to endothelium-denuded carotid arteries of apolipoprotein E-deficient mice; treatment reduced atherosclerotic neointimal area by 50% (p = 0.03). Thus, NPY variants associate with atherosclerosis in two independent datasets (with strong age-of-onset effects) and show allele-specific expression with NPY levels, while NPY receptor antagonism reduces atherosclerosis in mice. We conclude that NPY contributes to atherosclerosis pathogenesis.

Graft-Extrinsic Cells Predominate in Vein Graft Arterialization

Objective—Vein graft disease involves neointimal smooth muscle cells, the origins of which are unclear. This study sought to characterize and quantitate vein graft infiltration by cells extrinsic to the graft in a mouse model of vein graft disease. Methods and Results—Inferior vena cava-to-carotid artery interposition grafting between C57Bl/6 and congenic &bgr;-galactosidase–expressing ROSA26 mice was performed. Vein grafts were harvested 6 weeks postoperatively and stained with X-gal. More than 60% of neointimal cells derived from the recipient, and 50% of these cells expressed smooth muscle &agr;-actin. The distribution of donor and recipient-derived cells within this vein graft wall layer was distinctly focal, consistent with focal infiltration and expansion of progenitor cells. When bone marrow transplantation with congenic green fluorescent protein (GFP)-expressing cells was used in vein graft recipients 1 month before surgery, abundant GFP-expressing cells appeared in the media, but not the neointima, of mature grafts. Endothelial cells in mature grafts derived from graft-intrinsic and graft-extrinsic sources and were, in part, of bone marrow origin. Conclusions—Cells extrinsic to the graft, including bone marrow-derived cells, predominate during vein graft remodeling.

Expression of Tumor Necrosis Factor Receptor-1 in Arterial Wall Cells Promotes Atherosclerosis

Objective—Mechanisms by which tumor necrosis factor-α (TNF) contributes to atherosclerosis remain largely obscure. We therefore sought to determine the role of the arterial wall TNF receptor-1 (TNFR1) in atherogenesis. Methods and Results—Carotid artery-to-carotid artery interposition grafting was performed with tnfr1−/− and congenic (C57Bl/6) wild-type (WT) mice as graft donors, and congenic chow-fed apolipoprotein E-deficient mice as recipients. Advanced atherosclerotic graft lesions developed within 8 weeks, and had 2-fold greater area in WT than in tnfr1−/− grafts. While the prevalence of specific atheroma cells was equivalent in WT and tnfr1−/− grafts, the overall abundance of cells was substantially greater in WT grafts. WT grafts demonstrated greater MCP-1, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 expression at both early and late time points, and proliferating cell nuclear antigen expression at early time points. Aortic atherosclerosis was also reduced in 14-month-old apoe−/−/tnfr1−/− mice, as compared with cognate apoe−/− mice. In coculture with activated macrophages, smooth muscle cells expressing the TNFR1 demonstrated enhanced migration and reduced scavenger receptor activity. Conclusions—TNFR1 signaling, just in arterial wall cells, contributes to the pathogenesis of atherosclerosis by enhancing arterial wall chemokine and adhesion molecule expression, as well as by augmenting medial smooth muscle cell proliferation and migration.

β-Arrestins Regulate Atherosclerosis and Neointimal Hyperplasia by Controlling Smooth Muscle Cell Proliferation and Migration

Atherosclerosis and arterial injury-induced neointimal hyperplasia involve medial smooth muscle cell (SMC) proliferation and migration into the arterial intima. Because many 7-transmembrane and growth factor receptors promote atherosclerosis, we hypothesized that the multifunctional adaptor proteins &bgr;-arrestin1 and -2 might regulate this pathological process. Deficiency of &bgr;-arrestin2 in ldlr−/− mice reduced aortic atherosclerosis by 40% and decreased the prevalence of atheroma SMCs by 35%, suggesting that &bgr;-arrestin2 promotes atherosclerosis through effects on SMCs. To test this potential atherogenic mechanism more specifically, we performed carotid endothelial denudation in congenic wild-type, &bgr;-arrestin1−/−, and &bgr;-arrestin2−/− mice. Neointimal hyperplasia was enhanced in &bgr;-arrestin1−/− mice, and diminished in &bgr;-arrestin2−/− mice. Neointimal cells expressed SMC markers and did not derive from bone marrow progenitors, as demonstrated by bone marrow transplantation with green fluorescent protein-transgenic cells. Moreover, the reduction in neointimal hyperplasia seen in &bgr;-arrestin2−/− mice was not altered by transplantation with either wild-type or &bgr;-arrestin2−/− bone marrow cells. After carotid injury, medial SMC extracellular signal-regulated kinase activation and proliferation were increased in &bgr;-arrestin1−/− and decreased in &bgr;-arrestin2−/− mice. Concordantly, thymidine incorporation and extracellular signal-regulated kinase activation and migration evoked by 7-transmembrane receptors were greater than wild type in &bgr;-arrestin1−/− SMCs and less in &bgr;-arrestin2−/− SMCs. Proliferation was less than wild type in &bgr;-arrestin2−/− SMCs but not in &bgr;-arrestin2−/− endothelial cells. We conclude that &bgr;-arrestin2 aggravates atherosclerosis through mechanisms involving SMC proliferation and migration and that these SMC activities are regulated reciprocally by &bgr;-arrestin2 and &bgr;-arrestin1. These findings identify inhibition of &bgr;-arrestin2 as a novel therapeutic strategy for combating atherosclerosis and arterial restenosis after angioplasty.

Phosphorylation of the Platelet-derived Growth Factor Receptor-β and Epidermal Growth Factor Receptor by G Protein-coupled Receptor Kinase-2 MECHANISMS FOR SELECTIVITY OF DESENSITIZATION

Accumulating evidence suggests that receptor protein-tyrosine kinases, like the platelet-derived growth factor receptor-β (PDGFRβ) and epidermal growth factor receptor (EGFR), may be desensitized by serine/threonine kinases. One such kinase, G protein-coupled receptor kinase-2 (GRK2), is known to mediate agonist-dependent phosphorylation and desensitization of multiple heptahelical receptors. In testing whether GRK2 could phosphorylate and desensitize the PDGFRβ, we first found by phosphoamino acid analysis that cells expressing GRK2 could serine-phosphorylate the PDGFRβ in an agonist-dependent manner. Augmentation or inhibition of GRK2 activity in cells, respectively, reduced or enhanced tyrosine phosphorylation of the PDGFRβ but not the EGFR. Either overexpressed in cells or as a purified protein, GRK2 demonstrated agonist-promoted serine phosphorylation of the PDGFRβ and, unexpectedly, the EGFR as well. Because GRK2 did not phosphorylate a kinase-dead (K634R) PDGFRβ mutant, GRK2-mediated PDGFRβ phosphorylation required receptor tyrosine kinase activity, as does PDGFRβ ubiquitination. Agonist-induced ubiquitination of the PDGFRβ, but not the EGFR, was enhanced in cells overexpressing GRK2. Nevertheless, GRK2 overexpression did not augment PDGFRβ down-regulation. Like the vast majority of GRK2 substrates, the PDGFRβ, but not the EGFR, activated heterotrimeric G proteins allosterically in membranes from cells expressing physiologic protein levels. We conclude that GRK2 can phosphorylate and desensitize the PDGFRβ, perhaps through mechanisms related to receptor ubiquitination. Specificity of GRK2 for receptor protein-tyrosine kinases, expressed at physiologic levels, may be determined by the ability of these receptors to activate heterotrimeric G proteins, among other factors.

Overexpression of G Protein–Coupled Receptor Kinase-2 in Smooth Muscle Cells Attenuates Mitogenic Signaling via G Protein–Coupled and Platelet-Derived Growth Factor Receptors

BACKGROUND Neointimal hyperplasia involves activation of smooth muscle cells (SMCs) by several G protein-coupled receptor (GPCR) agonists, including endothelin-1, angiotensin II, thrombin, and thromboxane A(2). Signaling of many GPCRs is diminished by GPCR kinase-2 (GRK2). We therefore tested whether overexpression of GRK2 in SMCs could diminish mitogenic signaling elicited by agonists implicated in the pathogenesis of neointimal hyperplasia. METHODS AND RESULTS Overexpression of GRK2 was achieved in primary rabbit aortic SMCs with a recombinant adenovirus. Control SMCs were infected with an empty vector adenovirus. Inositol phosphate responses to endothelin-1, angiotensin II, thrombin agonist peptide, and platelet-derived growth factor (PDGF) were attenuated by 37% to 72% in GRK2-overexpressing cells (P<0.01), but the response to the thromboxane A(2) analogue U46619 was unaffected. GRK2 also inhibited SMC [(3)H]thymidine incorporation stimulated not only by these agonists (by 30% to 60%, P<0.01) but also by 10% FBS (by 35%, P<0. 05). However, GRK2 overexpression had no effect on epidermal growth factor-induced [(3)H]thymidine incorporation. Agonist-induced tyrosine phosphorylation of the PDGF-beta receptor, but not the epidermal growth factor receptor, was reduced in GRK2-overexpressing SMCs. GRK2 overexpression also reduced SMC proliferation in response to endothelin-1, PDGF, and 10% FBS by 62%, 51%, and 29%, respectively (P<0.01), without any effect on SMC apoptosis. CONCLUSIONS GRK2 overexpression diminishes SMC mitogenic signaling and proliferation stimulated by PDGF or agonists for several GPCRs. Gene transfer of GRK2 may therefore be therapeutically useful for neointimal hyperplasia.