Trypsin cleavage of the β-adrenergic receptor.

Am J Physiol Heart Circ Physiol

Department of Pharmacology, Columbia University, New York City, New York.

Published: March 2022

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

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8897006PMC
http://dx.doi.org/10.1152/ajpheart.00005.2022DOI Listing

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