Abstract
β-adrenergic receptors (βARs) are prototypical G-protein coupled receptors that play a pivotal role in sympathetic regulation. In heart cells, β 1 R signaling mediates a global cAMP response, regulating both L-type Ca 2+ channels in the sarcolemma/T-tubules (SL/TTs) and ryanodine receptors in the sarcoplasmic reticulum (SR). In contrast, β 2 AR mediates local cAMP signaling with little effect on the function of SR proteins. Accumulating evidence in pathological and transgenic models has suggested a functional complexity of β 2 AR beyond its regulation of L-type Ca 2+ influx. So we investigated the signaling relationship between β 1 ARs and β 2 ARs. Using whole-cell patch clamp combined with confocal calcium imaging, we simultaneously measured L-type Ca 2+ currents and Ca 2+ transients in adult cardiomyocytes. β 1 AR stimulation with norepinephrine or isoproterenol increased both Ca 2+ currents and Ca 2+ transients. β 2 AR stimulation with Zinterol or Salbutamol only increased Ca 2+ currents (onside compartmentalization) and surprisingly inhibited the ability of β 1 AR in upregulating Ca 2+ transients (offside compartmentalization). Further evidence showed that β 2 AR activation recruited G-protein coupled receptor kinase 2 (GRK2) to cell membrane, which phosphorylated the ‘SSES’ serine cluster on β 1 AR carboxyl terminal. Phosphorylated β 1 AR coupled to β-arrestin1 and further recruited phosphodiesterase-4, leading to localized cAMP degradation. Immunocytochemical and biochemical analysis showed the β 2 AR-mediated offside compartmentalization was confined to the nanoscale domain. A knock-in rat model harboring mutations of the three serine residues in ‘SSES’ cluster of the β 1 AR C-terminus, a component of the putative β-arrestin1 binding site and GRK2 phosphorylation site, eliminated the offside compartmentalization conferred by β 2 AR activation. This finding reveals a fundamental “negative feed-forward” mechanism that serves to avoid the cytotoxicity of circulating catecholamine and to sharpen the transient β 1 AR response of sympathetic excitation, providing many potential novel targets for drug discovery against cardiovascular diseases.
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