Trypsin cleavage of the β 1 -adrenergic receptor.

β 1 -Adrenergic receptors (β 1 ARs) are the principal mediators of catecholamine action in cardiomyocytes. We previously showed that β 1 ARs accumulate as both full-length and NH 2 -terminally truncated species in cells, that maturational processing of full-length β 1 ARs to an NH 2 -terminally truncated form is attributable to O -glycan-regulated proteolytic cleavage of the β 1 AR NH 2 -terminus at R 31  ↓ L 32 by ADAM17, and that NH 2 -terminally truncated β 1 ARs remain signaling competent but they acquire a distinct signaling phenotype. NH 2 -terminally truncated β 1 ARs differ from full-length β 1 ARs in their signaling bias to cAMP/PKA versus ERK pathways and only the NH 2 -terminally truncated form of the β 1 AR constitutively activates AKT and confers protection against doxorubicin-dependent apoptosis in cardiomyocytes. Since the R 31  ↓ L 32 sequence conforms to a trypsin consensus cleavage site, we used immunoblotting methods to test the hypothesis that β 1 ARs are also cleaved at R 31  ↓ L 32 by trypsin (an enzyme typically used to isolate cardiomyocytes from the intact ventricle). We show that full-length β 1 ARs are cleaved by trypsin and that trypsin cleaves the full-length β 1 AR NH 2 -terminus specifically at R 31  ↓ L 32 in CHO-Pro5 cells. Trypsin also cleaves β 1 ARs in cardiomyocytes, but at a second site that results in the formation of ∼40-kDa NH 2 -terminal and ∼30-kDa COOH-terminal fragments. The observation that cardiomyocyte β 1 ARs are cleaved by trypsin (a mechanism that constitutes a heretofore-unrecognized mechanism that would influence β 1 AR-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 β 1 AR responses derive from literature predicated on the assumption that β 1 ARs signal exclusively as full-length receptor proteins. However, we recently showed that β 1 ARs accumulate as both full-length and NH 2 -terminally truncated forms. This manuscript provides novel evidence that β 1 -adrenergic receptors can be cleaved by trypsin and that cell surface β 1 AR cleavage constitutes a heretofore unrecognized mechanism to alter catecholamine-dependent signaling responses.