The ability of morphine to alleviate pain is mediated through a heterotrimeric guanine nucleotide binding protein (G protein)-coupled heptahelical receptor (GPCR), the mu opioid receptor (muOR). The efficiency of GPCR signaling is tightly regulated and ultimately limited by the coordinated phosphorylation of the receptors by specific GPCR kinases and the subsequent interaction of the phosphorylated receptors with beta-arrestin 1 and beta-arrestin 2. Functional deletion of the beta-arrestin 2 gene in mice resulted in remarkable potentiation and prolongation of the analgesic effect of morphine, suggesting that muOR desensitization was impaired. These results provide evidence in vivo for the physiological importance of beta-arrestin 2 in regulating the function of a specific GPCR, the muOR. Moreover, they suggest that inhibition of beta-arrestin 2 function might lead to enhanced analgesic effectiveness of morphine and provide potential new avenues for the study and treatment of pain, narcotic tolerance, and dependence.

SummaryUsing a multistep polymerase chain reaction method, we have produced a construct in which a cDNA sequence encoding the extraceUular domain of the human 55-kD tumor necrosis factor (TNF) receptor is attached to a sequence encoding the Fc portion and hinge region of a mouse IgG1 heavy chain through an oligomer encoding a thrombin-sensitive peptide linker. This construct was placed downstream from a cytomegalovirus promoter sequence, and expressed in Chinese hamster ovary cells. A secreted protein, capable of binding TNF and inactivating it, was produced by the transfected cells. Molecular characterization revealed that this soluble version of the TNF receptor was dimeric. Moreover, the protein could be quantitatively cleaved by treatment with thrombin. However, the monovalent extracellular domain prepared in this way has a greatly reduced TNF inhibitory activity compared with that of the bivalent inhibitor. Perhaps because of its high affinity for TNF, the chimeric protein is far more effective as a TNF inhibitor than are neutralizing monodonal antibodies. This molecule may prove very useful as a reagent for the antagonism and assay of TNF and lymphotoxin from diverse species in health and disease, and as a means of deciphering the exact mechanism through which TNF interacts with the 55-kD receptor.T he recent cloning of the 55-kD (1-3) and 75-kD (3-5) TNF receptors has opened the way for further studies of TNF effects and signal transduction. Moreover, it appears that truncated receptor molecules, lacking the transmembrane or cytoplasmic domains, are capable of interacting with TNF, and therefore have been isolated from urine (6, 7) and serum (2) as TNF inhibitors. We considered that derivatives of such molecules might prove useful as antagonists of TNF action in vivo, as high affinity ligands to be applied as the basis of a more sensitive assay for TNF, and as reagents to be used in defining the molecular interaction between TNF and its receptor.Unfortunately, truncated forms of the TNF receptor are highly unstable in vivo, and therefore are poor substitutes for antibodies as a means of antagonizing TNF action in living animals. The naturally occurring TNF receptor fragments are univalent and therefore have an avidity that is effectively far lower than that of a bivalent ligand. The production of large quantities of a truncated receptor by recombinant means has, in our hands, been problematic, since the protein is produced in an inactive and insoluble form in bacteria (Peppel, K., and B. Beutler, unpublished observations). This difficulty may arise from the highly cysteine-rich structure of the receptor-binding domain (1, 2). When produced in roammalian cells through recombinant techniques, the soluble receptor fragment is active, but produced at low levels, and therefore difficult to purify (Beutler, B., and T. Brown, unpublished observations).To circumvent these problems, we have engineered a chimeric protein in which the extracellular domain of the TNF receptor, which normally engages the TNF molecu...

A chimeric protein capable of binding and neutralizing tumor necrosis factor (TNF) and lymphotoxin was expressed in mice transduced with a replication-incompetent adenoviral vector into which a TNF inhibitor gene had been engineered. Within 3 days following the injection of 109 infectious particles, the TNF inhibitor concentration exceeded 1 mg/ml of plasma; this level of expression was maintained for at least 4 weeks, and detectable TNF inhibitory activity was measured 6 weeks after injection of the recombinant virus.

Cardiovascular gene therapy is a novel approach to the treatment of diseases such as congestive heart failure (CHF). Gene transfer to the heart would allow for the replacement of defective or missing cellular proteins that may improve cardiac performance. Our laboratory has been focusing on the feasibility of restoring ␤-adrenergic signaling deficiencies that are a characteristic of chronic CHF. We have now studied isolated ventricular myocytes from rabbits that have been chronically paced to produce hemodynamic failure. We document molecular ␤-adrenergic signaling defects including down-regulation of myocardial ␤-adrenergic receptors (␤-ARs), functional ␤-AR uncoupling, and an upregulation of the ␤-AR kinase (␤ARK1). Adenoviral-mediated gene transfer of the human ␤ 2 -AR or an inhibitor of ␤ARK1 to these failing myocytes led to the restoration of ␤-AR signaling. These results demonstrate that defects present in this critical myocardial signaling pathway can be corrected in vitro using genetic modification and raise the possibility of novel inotropic therapies for CHF including the inhibition of ␤ARK1 activity in the heart.

Abstract-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 ␤-arrestin1 and -2 might regulate this pathological process. Deficiency of ␤-arrestin2 in ldlr Ϫ/Ϫ mice reduced aortic atherosclerosis by 40% and decreased the prevalence of atheroma SMCs by 35%, suggesting that ␤-arrestin2 promotes atherosclerosis through effects on SMCs. To test this potential atherogenic mechanism more specifically, we performed carotid endothelial denudation in congenic wild-type, ␤-arrestin1 Ϫ/Ϫ , and ␤-arrestin2 Ϫ/Ϫ mice. Neointimal hyperplasia was enhanced in ␤-arrestin1mice, and diminished in ␤-arrestin2 Ϫ/Ϫ 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 ␤-arrestin2 Ϫ/Ϫ mice was not altered by transplantation with either wild-type or ␤-arrestin2 Ϫ/Ϫ bone marrow cells. After carotid injury, medial SMC extracellular signal-regulated kinase activation and proliferation were increased in ␤-arrestin1Ϫ/Ϫ and decreased in ␤-arrestin2 Ϫ/Ϫ mice. Concordantly, thymidine incorporation and extracellular signal-regulated kinase activation and migration evoked by 7-transmembrane receptors were greater than wild type in ␤-arrestin1 Ϫ/Ϫ SMCs and less in ␤-arrestin2 Ϫ/Ϫ SMCs. Proliferation was less than wild type in ␤-arrestin2 Ϫ/Ϫ SMCs but not in ␤-arrestin2 Ϫ/Ϫ endothelial cells. We conclude that ␤-arrestin2 aggravates atherosclerosis through mechanisms involving SMC proliferation and migration and that these SMC activities are regulated reciprocally by ␤-arrestin2 and ␤-arrestin1. These findings identify inhibition of ␤-arrestin2 as a novel therapeutic strategy for combating atherosclerosis and arterial restenosis after angioplasty.

G protein-coupled receptor kinases (GRKs) 2 and 3 (␤-adrenergic receptor kinases 1 and 2 (␤ARK1 and -2)) mediate the agonist-dependent phosphorylation and uncoupling of many G protein-coupled receptors. These two members of the GRK family share a high degree of sequence homology and show overlapping patterns of substrate specificity in vitro. To define their physiological roles in vivo we have generated mice that carry targeted disruption of these genes. In contrast to GRK2-deficient mice, which die in utero (Jaber, M., Koch, W. J., Rockman, H., Smith, B., Bond, R. A., Sulik, K. K., Ross, J. JR., Lefkowitz, R. J. Caron, M. G., and Giros, B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12974 -12979), GRK3 deletion allows for normal embryonic and postnatal development. GRK3 is expressed to a high degree in the olfactory epithelium, where GRK2 is absent. Here we report that cilia preparations derived from GRK3-deficient mice lack the fast agonist-induced desensitization normally seen after odorant stimulation. Moreover, total second messenger (cAMP) generation in these cilia preparations following odorant stimulation is markedly reduced when compared with preparations from wildtype littermates. This reduction in the ability to generate cAMP is evident even in the presence of nonodorant receptor stimuli (GTP␥S and forskolin), suggesting a compensatory dampening of the G protein-adenylyl cyclase system in the GRK3 (؊/؊) mice in the olfactory epithelium. These findings demonstrate the requirement of GRK3 for odorant-induced desensitization of cAMP responses.Many G protein-coupled receptors (GPCRs) 1 show diminished ability to signal and couple to G proteins after prolonged or repeated agonist stimulation. This phenomenon, referred to as agonist-mediated desensitization, occurs very rapidly and is initiated via receptor phosphorylation by G protein-coupled receptor kinases (GRKs) that serve to uncouple the receptor from its G protein (1).Whereas the function of these proteins has been mostly studied in vitro and in tissue culture the physiological relevance of the mechanisms initiated by them have just begun to be explored. While there is some evidence of substrate specificity among the different members of the GRK family, most show activity toward a wide variety of agonist-occupied receptors in vitro. This, in addition to their ubiquitous tissue expression, has made it difficult to precisely determine the role of the GRKs in vivo. To clarify the physiological role of the individual members of this family, we have generated mice that carry targeted disruptions of the GRK2 or GRK3 (␤ARK2) genes. GRK2 deletion is embryonically lethal as homozygous mice die in utero before gestational day 15.5 of severe cardiac malformations (2). Whereas GRKs 2 and 3 show 81% amino acid identity (3) and an overlapping pattern of tissue expression (4), GRK3 apparently is not able to compensate for the loss of GRK2 in embryogenesis. In most tissues examined GRK2 is the predominant form. However, in the olfactory epithelium GRK2 is virtually a...

Accumulating evidence suggests that receptor protein-tyrosine kinases, like the platelet-derived growth factor receptor-␤ (PDGFR␤) 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 agonistdependent phosphorylation and desensitization of multiple heptahelical receptors. In testing whether GRK2 could phosphorylate and desensitize the PDGFR␤, we first found by phosphoamino acid analysis that cells expressing GRK2 could serine-phosphorylate the PDGFR␤ in an agonist-dependent manner. Augmentation or inhibition of GRK2 activity in cells, respectively, reduced or enhanced tyrosine phosphorylation of the PDGFR␤ but not the EGFR. Either overexpressed in cells or as a purified protein, GRK2 demonstrated agonist-promoted serine phosphorylation of the PDGFR␤ and, unexpectedly, the EGFR as well. Because GRK2 did not phosphorylate a kinase-dead (K634R) PDGFR␤ mutant, GRK2-mediated PDGFR␤ phosphorylation required receptor tyrosine kinase activity, as does PDGFR␤ ubiquitination. Agonist-induced ubiquitination of the PDGFR␤, but not the EGFR, was enhanced in cells overexpressing GRK2. Nevertheless, GRK2 overexpression did not augment PDGFR␤ down-regulation. Like the vast majority of GRK2 substrates, the PDGFR␤, 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 PDGFR␤, 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.

Our laboratory has been testing the hypothesis that genetic modulation of the ␤ -adrenergic signaling cascade can enhance cardiac function. We have previously shown that transgenic mice with cardiac overexpression of either the human ␤ 2 -adrenergic receptor ( ␤ 2 AR) or an inhibitor of the ␤ -adrenergic receptor kinase ( ␤ ARK), an enzyme that phosphorylates and uncouples agonist-bound receptors, have increased myocardial inotropy. We now have created recombinant adenoviruses encoding either the ␤ 2 AR (Adeno-␤ 2 AR) or a peptide ␤ ARK inhibitor (consisting of the carboxyl terminus of ␤ ARK1, Adeno-␤ ARKct) and tested their ability to potentiate ␤ -adrenergic signaling in cultured adult rabbit ventricular myocytes. As assessed by radioligand binding, Adeno-