Caveolar Localization Dictates Physiologic Signaling of β2-Adrenoceptors in Neonatal Cardiac Myocytes

Abstract

There is a growing body of evidence that G proteincoupled receptors function in the context of plasma membrane signaling compartments. These compartments may facilitate interaction between receptors and specific downstream signaling components while restricting access to other signaling molecules. We recently reported that ␤ 1 -and ␤ 2 -adrenergic receptors (AR) regulate the intrinsic contraction rate in neonatal mouse myocytes through distinct signaling pathways. By studying neonatal myocytes isolated from ␤ 1 AR and ␤ 2 AR knockout mice, we found that stimulation of the ␤ 1 AR leads to a protein kinase A-dependent increase in the contraction rate. In contrast, stimulation of the ␤ 2 AR has a biphasic effect on the contraction rate. The biphasic effect includes an initial protein kinase A-independent increase in the contraction rate followed by a sustained decrease in the contraction rate that can be blocked by pertussis toxin. Here we present evidence that caveolar localization is required for physiologic signaling by the ␤ 2 AR but not the ␤ 1 AR in neonatal cardiac myocytes. Evidence for ␤ 2 AR localization to caveolae includes co-localization by confocal imaging, co-immunoprecipitation of the ␤ 2 AR and caveolin 3, and co-migration of the ␤ 2 AR with a caveolin-3-enriched membrane fraction. The ␤ 2 AR-stimulated increase in the myocyte contraction rate is increased by ϳ2-fold and markedly prolonged by filipin, an agent that disrupts lipid rafts such as caveolae and significantly reduces co-immunoprecipitation of ␤ 2 AR and caveolin 3 and comigration of ␤ 2 AR and caveolin-3 enriched membranes. In contrast, filipin has no effect on ␤ 1 AR signaling. These observations suggest that ␤ 2 ARs are normally restricted to caveolae in myocyte membranes and that this localization is essential for physiologic signaling of this receptor subtype.Catecholamines act through cardiac ␤-adrenergic receptors (␤ARs) 1 to modulate heart rate and contractility. Three ␤AR subtypes have been cloned (␤ 1 AR, ␤ 2 AR, and ␤ 3 AR). ␤ 1 AR and ␤ 2 AR are the primary subtypes responsible for cardiac response to catecholamines. ␤ 1 AR and ␤ 2 AR are also pharmacologically more similar to each other than they are to the ␤ 3 AR. The close structural and functional properties of ␤ 1 AR and ␤ 2 AR are paradigmatic of many other G protein-coupled receptor families in which two or more receptor subtypes respond to the same hormone or neurotransmitter and couple to the same effector systems. Although ␤ 1 AR and ␤ 2 AR have very similar signaling properties when expressed in undifferentiated cell lines (1), there is a growing body of experimental evidence that suggests that they have different signaling properties in regulating cardiac function. The ␤ 1 AR knockout (␤ 1 AR-KO) mice lack the normal chronotropic and inotropic responses to the non-selective agonist isoproterenol (2). Thus, in the murine heart, ␤ 2 ARs play no significant role in controlling heart rate and contractility. ␤ 2 AR knockout (␤ 2 AR-KO) mice have normal inotropic and chr...

Regulation of cardiac contractility by Rab4-modulated β2-adrenergic receptor recycling

“…This finding is consistent with previously described agonist-induced caveolar targeting of m2 muscarinic and kinin B1 receptors (34,35) but differs from prior descriptions of cardiomyocyte ␤2AR as being located entirely in the caveolar-rich fractions of cultured neonatal rat cardiomyocytes (29,31). Although the h-␤2AR transgenic model may, like any overexpression or transfection system, generate nonphysiological results, other possible explanations for these differences include species differences between rodent and human ␤2AR, developmental differences between neonatal and adult cardiomyocytes (25), the total absence of intrinsic catecholamine stimulation and contractile activity in quiescent tissue culture, and altered ␤2AR localization after enzymatic cardiomyocyte isolation (25).…”

“…In h-␤2AR-overexpressing mice representing the normal pattern of ␤2AR compartmentation, there was highly defined labeling of sarcolemma and transverse tubular structures (Fig. 4A), consistent with sarcolemmal localization (31). In contrast, Rab4 S27N͞h-␤2AR cardiomyocytes exhibited granular cytoplasmic staining and coarse labeling of sarcolemma and transverse tubules, suggesting subsarcolemmal vesicular localization of receptors (Fig.…”

Integrins regulate opioid receptor signaling in trigeminal ganglion neurons

“…For example, association with caveolae regulates the relative efficiency of coupling of the ß2-adrenergic receptor to Gαs vs. Gαi (Xiang et al, 2002). In our study, a MOR agonist (DAMGO) significantly decreased cAMP accumulation in PGE 2 -stimulated bradykinin-primed trigeminal ganglion neurons, but significantly increased cAMP accumulation in the presence of β1 integrin antagonism.…”

Phosphodiesterase 4D is required for β 2 adrenoceptor subtype-specific signaling in cardiac myocytes

“…Myocyte cultures were maintained in DMEM containing 10% Nu serum, 10% bovine fetal serum, and 1ϫ Gentamycin. The culture media were changed every 24 h. Measurement of spontaneous contraction rate was carried out as described (5). Stimulation on ␤ARs on mouse neonatal cardiac myocytes leads to a robust increase in contraction rate with an average increase from Ϸ220 beats per min to Ϸ270 beats per min.…”