␤ 1 -and ␤ 2 -adrenergic receptors (␤ 1 AR and ␤ 2 AR) are co-expressed in numerous tissues where they play a central role in the responses of various organs to sympathetic stimulation. Although the two receptor subtypes share some signaling pathways, each has been shown to have specific signaling and regulatory properties. Given the recent recognition that many G protein-coupled receptors can form homo-and heterodimers, the present study was undertaken to determine whether the ␤ 1 AR and ␤ 2 AR can form dimers in cells and, if so, to investigate the potential functional consequences of such heterodimerization. Using co-immunoprecipitation and bioluminescence resonance energy transfer, we show that ␤ 1 AR and ␤ 2 AR can form heterodimers in HEK 293 cells co-expressing the two receptors. Functionally, ␤-adrenergic stimulated adenylyl cyclase activity was found to be identical in cells expressing ␤ 1 AR, ␤ 2 AR, or both receptors at similar levels, indicating that heterodimerization did not affect this signaling pathway. When considering ERK1/2 MAPK activity, a significant agonistpromoted activation was detected in ␤ 2 AR-but not ␤ 1 AR-expressing cells. Similarly to what was observed in cells expressing the ␤ 1 AR alone, no ␤-adrenergic stimulated ERK1/2 phosphorylation was observed in cells co-expressing the two receptors. A similar inhibition of agonist-promoted internalization of the ␤ 2 AR was observed upon co-expression of the ␤ 1 AR, which by itself internalized to a lesser extent. Taken together, our data suggest that heterodimerization between ␤ 1 AR and ␤ 2 AR inhibits the agonist-promoted internalization of the ␤ 2 AR and its ability to activate the ERK1/2 MAPK signaling pathway.

These results highlight differential subcellular localization for betaAR subtypes and indicate that betaAR may have specific roles in regulating nuclear function in cardiomyocytes.

Abstract-Intermolecular interactions between members of both similar and divergent G protein-coupled receptor subfamilies have been shown in various experimental systems. Here, we demonstrate heterodimerization of predominant ␤-adrenergic receptor (␤AR) subtypes expressed in the heart, ␤ 1 AR, and ␤ 2 AR, and its physiological relevance. In intact adult-mouse cardiac myocytes lacking native ␤ 1 AR and ␤ 2 AR, coexpression of both ␤AR subtypes led to receptor heterodimerization, as evidenced by their coimmunoprecipitation, colocalization at optical resolution, and markedly increased binding affinity for subtype-selective ligands. As a result, the dose-response curve of myocyte contraction to ␤AR agonist stimulation with isoproterenol (ISO) was shifted leftward by Ϸ1.5 orders of magnitude, and the response of cellular cAMP formation to ISO was enhanced concomitantly, indicating that intermolecular interactions of ␤AR subtypes resulted in sensitization of these receptors in response to agonist stimulation. In contrast, the presence of ␤ 1 AR greatly suppressed ligand-independent spontaneous activity of coexisting ␤ 2 ARs. Thus, heterodimerization of ␤ 1 AR and ␤ 2 AR in intact cardiac myocytes creates a novel population of ␤ARs with distinct functional and pharmacological properties, resulting in enhanced signaling efficiency in response to agonist stimulation while silencing ligandindependent receptor activation, thereby optimizing ␤-adrenergic modulation of cardiac contractility.

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