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.