Trypsin cleavage of the β1-adrenergic receptor.

β1-Adrenergic receptors (β1ARs) are the principal mediators of catecholamine action in cardiomyocytes. We previously showed that β1ARs accumulate as both full-length and NH2-terminally truncated species in cells, that maturational processing of full-length β1ARs to an NH2-terminally truncated form is attributable to O-glycan-regulated proteolytic cleavage of the β1AR NH2-terminus at R31 ↓ L32 by ADAM17, and that NH2-terminally truncated β1ARs remain signaling competent but they acquire a distinct signaling phenotype. NH2-terminally truncated β1ARs differ from full-length β1ARs in their signaling bias to cAMP/PKA versus ERK pathways and only the NH2-terminally truncated form of the β1AR constitutively activates AKT and confers protection against doxorubicin-dependent apoptosis in cardiomyocytes. Since the R31 ↓ L32 sequence conforms to a trypsin consensus cleavage site, we used immunoblotting methods to test the hypothesis that β1ARs are also cleaved at R31 ↓ L32 by trypsin (an enzyme typically used to isolate cardiomyocytes from the intact ventricle). We show that full-length β1ARs are cleaved by trypsin and that trypsin cleaves the full-length β1AR NH2-terminus specifically at R31 ↓ L32 in CHO-Pro5 cells. Trypsin also cleaves β1ARs in cardiomyocytes, but at a second site that results in the formation of ∼40-kDa NH2-terminal and ∼30-kDa COOH-terminal fragments. The observation that cardiomyocyte β1ARs are cleaved by trypsin (a mechanism that constitutes a heretofore-unrecognized mechanism that would influence β1AR-signaling responses) suggests that studies that use standard trypsin-based procedures to isolate adult cardiomyocytes from the intact ventricle should be interpreted with caution.NEW & NOTEWORTHY Current concepts regarding the molecular basis for β1AR responses derive from literature predicated on the assumption that β1ARs signal exclusively as full-length receptor proteins. However, we recently showed that β1ARs accumulate as both full-length and NH2-terminally truncated forms. This manuscript provides novel evidence that β1-adrenergic receptors can be cleaved by trypsin and that cell surface β1AR cleavage constitutes a heretofore unrecognized mechanism to alter catecholamine-dependent signaling responses.