Recent data have demonstrated that caveolin, a major structural protein of caveolae, negatively regulates signaling molecules localized to caveolae. The interaction of caveolin with several caveolae-associated signaling proteins is mediated by the binding of the scaffolding region of caveolin to a hydrophobic amino acid-containing region within the regulated proteins. The presence of a similar motif within the insulin receptor kinase prompted us to investigate the caveolar localization and regulation of the insulin receptor by caveolin. We found that overexpression of caveolin-3 augmented insulinstimulated phosphorylation of insulin receptor substrate-1 in 293T cells but not the phosphorylation of insulin receptor. Peptides corresponding to the scaffolding domain of caveolin potently stimulated insulin receptor kinase activity toward insulin receptor substrate-1 or a Src-derived peptide in vitro and in a caveolin subtype-dependent fashion. Peptides from caveolin-2 exhibited no effect, whereas caveolin-1 and -3 stimulated activity 10-and 17-fold, respectively. Peptides which increased insulin receptor kinase activity did so without affecting insulin receptor auto-phosphorylation. Furthermore, the insulin receptor bound to immobilized caveolin peptides, and this binding was inhibited in the presence of free caveolin-3 peptides. Thus, we have identified a novel mechanism by which the insulin receptor is bound and activated by specific caveolin subtypes. Furthermore, these data define a new role for caveolin as an activator of signaling.

Various neurotransmitters, such as dopamine, stimulate adenylyl cyclase to produce cAMP, which regulates neuronal functions. Genetic disruption of the type 5 adenylyl cyclase isoform led to a major loss of adenylyl cyclase activity in a striatum-specific manner with a small increase in the expression of a few other adenylyl cyclase isoforms. D1 dopaminergic agonist-stimulated adenylyl cyclase activity was attenuated, and this was accompanied by a decrease in the expression of the D1 dopaminergic receptor and G s ␣. D2 dopaminergic agonist-mediated inhibition of adenylyl cyclase activity was also blunted. Type 5 adenylyl cyclase-null mice exhibited Parkinsonian-like motor dysfunction, i.e. abnormal coordination and bradykinesia detected by Rotarod and pole test, respectively, and to a lesser extent locomotor impairment was detected by open field tests. Selective D1 or D2 dopaminergic stimulation improved some of these disorders in this mouse model, suggesting the partial compensation of each dopaminergic receptor signal through the stimulation of remnant adenylyl cyclase isoforms. These findings extend our knowledge of the role of an effector enzyme isoform in regulating receptor signaling and neuronal functions and imply that this isoform provides a site of convergence of both D1 and D2 dopaminergic signals and balances various motor functions.

Adenylyl cyclase, a major target enzyme of ␤-adrenergic receptor signals, is potently and directly inhibited by P-site inhibitors, classic inhibitors of this enzyme, when the enzyme catalytic activity is high. Unlike ␤-adrenergic receptor antagonists, this is a non-or uncompetitive inhibition with respect to ATP. We have examined whether we can utilize this enzymatic property to regulate the effects of ␤-adrenergic receptor stimulation differentially. After screening multiple new and classic compounds, we found that some compounds, including 1R,4R-3-(6-aminopurin-9-yl)-cyclopentanecarboxylic acid hydroxyamide, potently inhibited type 5 adenylyl cyclase, the major cardiac isoform, but not other isoforms. In normal mouse cardiac myocytes, contraction induced by low ␤-adrenergic receptor stimulation was poorly inhibited with this compound, but the induction of cardiac myocyte apoptosis by high ␤-adrenergic receptor stimulation was effectively prevented by type 5 adenylyl cyclase inhibitors. In contrast, when cardiac myocytes from type 5 adenylyl cyclase knock-out mice were examined, ␤-adrenergic stimulation poorly induced apoptosis. Our data suggest that the inhibition of ␤-adrenergic signaling at the level of the type 5 adenylyl cyclase isoform by P-site inhibitors may serve as an effective method to prevent cardiac myocyte apoptosis induced by excessive ␤-adrenergic stimulation without deleterious effect on cardiac myocyte contraction.Adrenergic stimulation via the sympathetic nervous system is a major mechanism to regulate cardiac function (1). Norepinephrine released from the synaptic terminal binds to ␤-adrenergic receptors (␤-AR), 1 leading to the activation of adenylyl cyclase (AC) and thus the production of cAMP, which initiates a cascade of protein phosphorylation and regulation of many key signaling elements involved in muscle contraction and calcium handling. The immediate outcome of this stimulation is increased cardiac contractility and output. Pharmacological inhibition of this pathway, by the use of ␤-AR antagonists, has been widely utilized to attenuate cardiac function in the clinical setting of treating high blood pressure for several decades. Recently, ␤-AR antagonists have been used in the treatment of heart failure, i.e. in hearts with deteriorated cardiac function. This paradoxical usage of ␤-AR antagonists in heart failure has been rationalized by the protection of the heart from excessive sympathetic stimulation. Under this pathological condition, it is well known that the sympathetic activity is increased markedly (2-4), leading to the remodeling of the heart (5) and, more importantly, the induction of cardiac myocyte apoptosis, as demonstrated by both in vivo and in vitro studies (6, 7). However, a major problem in introducing ␤-AR antagonist therapy is the worsening of cardiac function (8).It is believed that regulating ␤-adrenergic signaling at the level of AC, instead of the receptor, may serve as an alternative to the common ␤-AR regulating therapy, which is most exemplified by direct ...

The recently identified endogenous peptide apelin and its specific apelin receptor (APJ) are currently being considered as potential regulators in vascular tissue. Previously, we reported apelin mediates phosphorylation of myosin light chain and elicits vasoconstriction in vascular smooth muscle. In this study, physiological roles of the apelin-APJ system were investigated on atherosclerosis. In APJ and apolipoprotein E double-knockout (APJ ؊/؊ ApoE ؊/؊ ) mice fed a high-cholesterol diet, atherosclerotic lesions were dramatically reduced when compared with APJ Apelin receptor (APJ) is a G-protein-coupled receptor with seven transmembrane domains, and its endogenous ligand, apelin has been recently identified.1,2 The structures of APJ and apelin are highly conserved among species, and both are highly expressed in the cardiovascular system. 3,4 In the vascular system, APJ and apelin are known to be expressed in endothelium and vascular smooth muscle cells (VSMCs). Histological studies in rat show that the VSMCs of the medial layer of the aorta and pulmonary artery display intense staining for APJ-like immunoreactivity. 4 The vascular actions of apelin-APJ system may be complex. Under physiological conditions, the apelin-APJ system shows transient hypotension. The baseline blood pressure of APJ and angiotensin-type 1 receptor doubleknockout mice was significantly elevated compared with that of angiotensin-type 1 receptor knockout mice, 5 although APJ knockout (APJ Ϫ/Ϫ ) mice did not show any significant changes in cardiovascular parameters. In spontaneously hypertensive rats, APJ and apelin expression in both heart and aorta were markedly depressed compared with Wistar-Kyoto rats. 6 In aortae from type 2 diabetic db/db mice, APJ and apelin expression were

The renin-angiotensin system in the kidney plays a critical role in the regulation of renal hemodynamics and sodium handling through the activation of vascular, glomerular and tubular angiotensin II type 1 (AT1) receptor-mediated signaling. We previously cloned a molecule that specifically bound to the AT1 receptor and modulated AT1 receptor signaling in vitro, which we named ATRAP (for AT1 receptor-associated protein). The purpose of this study is to analyze the renal distribution of ATRAP and to examine whether ATRAP is co-expressed with the AT1 receptor in the mouse kidney. We performed in situ hybridization, Western blot analysis, and immunohistochemistry to investigate the expression of ATRAP mRNA and protein in the mouse kidney. The results of Western blot analysis revealed the ATRAP protein to be abundantly expressed in the kidney. Employing in situ hybridization and immunohistochemistry, we found that both ATRAP mRNA and the protein were widely distributed along the renal tubules from Bowman's capsules to the inner medullary collecting ducts. ATRAP mRNA was also detected in the glomeruli, vasculature, and interstitial cells. In all tubular cells, the ATRAP protein colocalized with the AT1 receptor. Finally, we found that the dietary salt depletion significantly decreased the renal expression of ATRAP as well as AT1 receptor. These findings show ATRAP to be abundantly and broadly distributed in nephron segments where the AT1 receptor is expressed. Furthermore, this is the first report demonstrating a substantial colocalization of ATRAP and AT1 receptor in vivo.