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...

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The b 1 -adrenergic receptor (b 1 AR) is the predominant bAR in the heart and is the main target for b-adrenergic antagonists, widely used in the treatment of cardiovascular diseases. Previously, we have shown that the human (h) b 1 AR is cleaved in its N terminus by a metalloproteinase, both constitutively and in a receptor activation-dependent manner. In this study, we investigated the specific events involved in b 1 AR regulation, focusing on the effects of long-term treatment with b-adrenergic ligands on receptor processing in stably transfected human embryonic kidney 293 i cells. The key findings were verified using the transiently transfected hb 1 AR and the endogenously expressed receptor in neonatal rat cardiomyocytes. By using flow cytometry and Western blotting, we demonstrated that isoproterenol, S-propranolol, CGP-12177 [4-[3-[(1,1-dimethylethyl)amino]2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one], pindolol, and timolol, which displayed agonistic properties toward the b 1 AR in either the adenylyl cyclase or the mitogen-activated protein kinase signaling pathways, induced cleavage of the mature cell-surface receptor. In contrast, metoprolol, bisoprolol, and CGP-20712 [1-[2-((3-carbamoyl-4-hydroxy) phenoxy)ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl) phenoxy]-2-propanol], which showed no agonistic activity, had only a marginal or no effect. Importantly, the agonists also stabilized intracellular receptor precursors, possibly via their pharmacological chaperone action, and they stabilized the receptor in vitro. The opposing effects on the two receptor forms thus led to an increase in the amount of cleaved receptor fragments at the plasma membrane. The results underscore the pluridimensionality of b-adrenergic ligands and extend this property from receptor activation and signaling to the regulation of b 1 AR levels. This phenomenon may contribute to the exceptional resistance of b 1 ARs to downregulation and tendency toward upregulation following long-term ligand treatments.

Increasing evidence suggests that the folding and maturation of monomeric proteins and assembly of multimeric protein complexes in the endoplasmic reticulum (ER) may be inefficient not only for mutants that carry changes in the primary structure but also for wild type proteins. In the present study, we demonstrate that the rat luteinizing hormone receptor, a G protein-coupled receptor, is one of these proteins that matures inefficiently and appears to be very prone to premature degradation. A substantial portion of the receptors in stably transfected human embryonic kidney 293 cells existed in immature form of M r 73,000, containing high mannose-type N-linked glycans. In metabolic pulse-chase studies, only ϳ20% of these receptor precursors were found to gain hormone binding ability and matured to a form of M r 90,000, containing bi-and multiantennary sialylated N-linked glycans. The rest had a propensity to form disulfide-bonded complexes with a M r 120,000 protein in the ER membrane and were eventually targeted for degradation in proteasomes. The number of membrane-bound receptor precursors increased when proteasomal degradation was inhibited, and no cytosolic receptor forms were detected, suggesting that retrotranslocation of the misfolded/incompletely folded receptors is tightly coupled to proteasomal function. Furthermore, a proteasomal blockade was found to increase the number of receptors that were capable of hormone binding. Thus, these results raise the interesting possibility that luteinizing hormone receptor expression at the cell surface may be controlled at the ER level by regulating the number of newly synthesized proteins that will mature and escape the ER quality control and premature degradation.

Background: The beta-1 adrenergic receptor (β 1 AR) plays a fundamental role in the regulation of cardiovascular functions. It carries a nonsynonymous single nucleotide polymorphism in its carboxyl terminal tail (Arg389Gly), which has been shown to associate with various echocardiographic parameters linked to left ventricular hypertrophy (LVH). Diabetes mellitus (DM), on the other hand, represents a risk factor for LVH. We investigated the possible association between the Arg389Gly polymorphism and LVH among non-diabetic and diabetic acute myocardial infarction (AMI) survivors. Methods: The study population consisted of 452 AMI survivors, 20.6% of whom had diagnosed DM. Left ventricular parameters were measured with two-dimensional guided M-mode echocardiography 2-7 days after AMI, and the Arg389Gly polymorphism was determined using a polymerase chain reaction-restriction fragment length polymorphism assay. Results: The Arg389 homozygotes in the whole study population had a significantly increased left ventricular mass index (LVMI) when compared to the Gly389 carriers (either Gly389 homozygotes or Arg389/Gly389 heterozygotes) [62.7 vs. 58.4, respectively (p = 0.023)]. In particular, the Arg389 homozygotes displayed thicker diastolic interventricular septal (IVSd) measures when compared to the Gly389 carriers [13.2 vs. 12.3 mm, respectively (p = 0.004)]. When the euglycemic and diabetic patients were analyzed separately, the latter had significantly increased LVMI and diastolic left ventricular posterior wall (LVPWd) values compared to the euglycemic patients [LVMI = 69.1 vs. 58.8 (p = 0.001) and LVPWd = 14.2 vs. 12.3 mm (p < 0.001), respectively]. Furthermore, among the euglycemic patients, the Arg389 homozygotes displayed increased LVMI and IVSd values compared to the Gly389 carriers [LVMI = 60.6 vs. 56.3, respectively (p = 0.028) and IVSd = 13.1 vs. 12.0 mm, respectively (p = 0.001)]. There was no difference in the LVMI and IVSd values between the diabetic Arg389 homozygotes and Gly389 carriers.