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Human beta1-adrenergic receptor is subject to constitutive and regulated N-terminal cleavage.

ABSTRACT: The beta(1)-adrenergic receptor (beta(1)AR) is the predominant betaAR 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 beta(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 mucin-type 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 betaAR 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 beta(1)ARs.

SUBMITTER: Hakalahti AE

PROVIDER: S-EPMC2937912 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Human beta1-adrenergic receptor is subject to constitutive and regulated N-terminal cleavage.

Hakalahti Anna E AE Vierimaa Miia M MM Lilja Minna K MK Kumpula Esa-Pekka EP Tuusa Jussi T JT Petäjä-Repo Ulla E UE

The Journal of biological chemistry 20100629 37

The beta(1)-adrenergic receptor (beta(1)AR) is the predominant betaAR 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 beta(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 ...[more]

PMID: 20587416

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Project description:BACKGROUND: Inhibition of proteolytic MMP activity could be a therapeutic approach to prevent ventricular dilatation by diminishing collagen matrix turnover and interstitial fibrosis. We investigated the time-course of MMP/TIMP activity during transition from hypertrophy to ventricular dilatation in transgenic mice with myocyte overexpression of the human beta1-adrenergic receptor (beta1TG). These beta1TG mice were studied at 3 (normal function), 5 (hypertrophy) and 12 (ventricular dilatation) months of age compared to age-matched controls (WT). METHODS: Picro Sirius red staining and real-time PCR were performed for total collagen and for collagen type I and III quantification, respectively. MMP-activity assays (zymography), immunoblotting and real-time PCR experiments were done for gelatinase- (MMP-2, -9), collagenase- (MMP-1, -13), membrane-type MMP- (MT1- MMP; MMP-14) and TIMP expression measurements. To investigate beta1-integrin activity, integrin-linked kinase (ILK) expression was measured by immunoblotting. RESULTS: Compared to WT with normal cardiac function, interstitial collagen type I and III mRNA and protein expression increased 3.6-fold in beta1TG at 5 months of age with moderate fibrosis and cardiomyocyte hypertrophy and 17-fold in beta1TG at 12 months of age with severe fibrosis and ventricular dilatation. Protein expression of the collagenases MMP-1 and -13 as well as the gelatinase proMMP-2 increased in the beta1TG group with cardiac hypertrophy. Maximal activity of the gelatinase MMP-2 (3.5-fold vs.WT) was measured in beta1TG at 12 months of age with severe fibrosis and ventricular dilatation, accompanied by coexpression of MT1- MMP (3.8-fold vs.WT) colocalized to the cell membranes. CONCLUSION: These data provide evidence that sympathetic overactivation can trigger interstitial matrix remodeling and fibrosis by induction of MMP/TIMP activity. In particular gelatinolytic MMP-2 activity accompanies ventricular dilatation and the development of heart failure.

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Human beta1-adrenergic receptor is subject to constitutive and regulated N-terminal cleavage.

Publications

Human beta1-adrenergic receptor is subject to constitutive and regulated N-terminal cleavage.

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