Human β1-Adrenergic Receptor Is Subject to Constitutive and Regulated N-terminal Cleavage

Abstract

The ␤ 1 -adrenergic receptor (␤ 1 AR) is the predominant ␤AR in the heart, mediating the catecholamine-stimulated increase in cardiac rate and force of contraction. Regulation of this important G protein-coupled receptor is nevertheless poorly understood. We describe here the biosynthetic profile of the human ␤ 1 AR and reveal novel features relevant to its regulation using an inducible heterologous expression system in HEK293 i cells. Metabolic pulse-chase labeling and cell surface biotinylation assays showed that the synthesized receptors are efficiently and rapidly transported to the cell surface. The N terminus of the mature receptor is extensively modified by sialylated mucintype O-glycosylation in addition to one N-glycan attached to Asn 15 . Furthermore, the N terminus was found to be subject to limited proteolysis, resulting in two membrane-bound C-terminal fragments. N-terminal sequencing of the fragments identified two cleavage sites between Arg 31 and Leu 32 and Pro 52 and Leu 53 , which were confirmed by cleavage site and truncation mutants. Metalloproteinase inhibitors were able to inhibit the cleavage, suggesting that it is mediated by a matrix metalloproteinase or a disintegrin and metalloproteinase (ADAM) family member. Most importantly, the N-terminal cleavage was found to occur not only in vitro but also in vivo. Receptor activation mediated by the ␤AR agonist isoproterenol enhanced the cleavage in a concentration-and time-dependent manner, and it was also enhanced by direct stimulation of protein kinase C and adenylyl cyclase. Mutation of the Arg 31 -Leu 32 cleavage site stabilized the mature receptor. We hypothesize that the N-terminal cleavage represents a novel regulatory mechanism of cell surface ␤ 1 ARs.The ␤ 1 -adrenergic receptor (␤ 1 AR) 3 is one of the three ␤AR subtypes that are activated by the endogenous catecholamines adrenaline and noradrenaline (1). These receptors belong to the G protein-coupled receptor (GPCR) family, one of the largest membrane protein families involved in cellular signaling (2, 3).The ␤ 1 AR is the predominant ␤AR subtype in the heart, mediating the increase in cardiac rate and force of contraction (4, 5). This makes it the most important target receptor for the ␤-blockers that are used to treat common cardiac diseases such as chronic heart failure, coronary artery disease, hypertension, and arrhythmias. The mechanisms that regulate human ␤ 1 AR (h␤ 1 AR) are therefore of considerable interest.The h␤ 2 AR is one of the most extensively studied GPCRs, but much less is known about h␤ 1 AR. The suggestion that it may be more resistant to agonist-mediated desensitization (6), internalization (7-12), and down-regulation (7, 10, 13, 14) could indicate that the two receptors are regulated by distinct mechanisms. Their ligand-binding sites are well conserved, but the overall homology of the two ␤ARs is only 54% (15). The most diverse regions are the intervening loops that connect the transmembrane domains, the extracellular N terminus and the intracellular C terminu...

Recombinant Human Matrix Metalloproteinase‐2 Impairs Cardiovascular β‐Adrenergic Responses

“…These findings clearly suggest that MMP-2 may affect the responsiveness of b 1 -adrenoreceptors. Although we have not determined molecular mechanisms in the present study, our findings are consistent with a recent report showing that GM6001 (a non-specific MMP inhibitor) prevented the cleavage of the N-terminus of the b 1 -adrenergic receptor [24]. Giving further support to our findings, increased MMP-2 expression impaired contraction and reduced the responses to inotropic stimulation in transgenic mice overexpressing MMP-2 [37,38].…”

“…However, this suggestion may not be true in animal models of disease conditions. Matrix metalloproteinases, particularly MMP-2, are known to affect b 1 -and b 2 -adrenoreceptors [21][22][23][24]. The significant attenuation in dobutamine-induced reduction in SVRI after the infusion of rhMMP-2 is consistent with impaired b 2 -adrenoreceptors-mediated vasodilation possibly resulting of enhanced MMP-2 activity.…”

“…Supporting their findings, another study showed that MMP-2 is involved in the proteolysis of the extracellular domain of b 2 -adrenoreceptors in kidney from spontaneously hypertensive rats [23]. Furthermore, Hakalahti et al [24] showed that GM6001 (a non-specific MMP inhibitor) prevented the cleavage of the N-terminus of the b 1 -adrenergic receptor.…”

The Parkinson's-disease-associated receptor GPR37 undergoes metalloproteinase-mediated N-terminal cleavage and ectodomain shedding

“…In addition to ET B R, the cleavage of β 1 adrenergic and V 2 vasopressin receptors has been reported to occur in an activationdependent manner following ligand binding (Kojro and Fahrenholz, 1995;Hakalahti et al, 2010Hakalahti et al, , 2013. A similar mechanism has been implicated for TSHR (Latif et al, 2004;Kaczur et al, 2007), although conflicting information exists (Chazenbalk et al, 2004).…”

Global Mapping of O-Glycosylation of Varicella Zoster Virus, Human Cytomegalovirus, and Epstein-Barr Virus

“…Based on the relatively short N-terminal regions of these receptors, it is quite unlikely for them to be involved in dimerization, as seen for GPCRs bearing large extracellular domains (108). However, glycosylation could potentially modulate ligand binding or limited proteolysis-associated receptor turnover, as hypothesized for the N terminus of the ␤1-adrenergic receptor (109). Among other specific functions, O-linked glycans may contribute to transport and stable cell surface expression of viral proteins.…”