Inefficient Maturation of the Rat Luteinizing Hormone Receptor

Pietilä, E. Maritta; Tuusa, Jussi; Apaja, Pirjo M.; Aatsinki, Jyrki T.; Hakalahti, Anna E.; Rajaniemi, Hannu; Petäjä-Repo, Ulla E.

doi:10.1074/jbc.m413815200

Cited by 73 publications

(15 citation statements)

References 71 publications

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“…Maturation of the h␤ 1 AR Is Efficient and Displays Relatively Fast Kinetics-The low relative amount of h␤ 1 AR precursors in HEK293 i cells suggests that synthesized receptors mature efficiently, a finding that is in contrast to those obtained in the same cellular background for two other GPCRs in the rhodopsin family, namely the human ␦-opioid (24, 26) and the ratluteinizing hormone receptors (21). Thus, to investigate h␤ 1 AR processing and maturation in more detail, cells were subjected to metabolic labeling.…”

Section: Ar Is Expressed As Two Full-length Receptor Forms-contrasting

confidence: 47%

“…The N-terminally truncated h␤ 1 AR constructs were cloned by PCR amplification from cDNAs encoding Met and Met , using the primer pairs 5Ј-GTCGAAGCTTATGCTGCTGGTG-CCCGCG-3Ј/5Ј-GTCGAAGCTTATGCTGTCTCAGCAGTG-GACAG-3Ј, and 5Ј-CGCCGGCCTAGGCACCTTGGATTCC-GAGGC-3Ј, followed by digestion with HindIII and AvrII (New England Biolabs), and ligation into the pFT-SMMF vector digested with the same enzymes. The pFT-SMMF vector was modified from the pcDNA5/FRT/TO vector (Invitrogen) as described previously (21). The DNA construct for the N-terminally HA-tagged h␤ 1 AR in pcDNA3 (Invitrogen) was obtained from Professor M. Bouvier and has been described elsewhere (22).…”

Section: Methodsmentioning

confidence: 99%

“…ECL or ECL Plus detection reagents (GE Healthcare) were used to reveal the blots. Gels containing radiolabeled samples were treated for fluorography as described previously (21). Exposed films were scanned with the Umax PowerLook 1120 color scanner and Image Master 2D Platinum 6.0 software, and the data were quantified and analyzed as described previously (21).…”

Section: Sds-page Western Blotting and Fluorography-samplesmentioning

confidence: 99%

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

Hakalahti¹,

Vierimaa

Lilja

et al. 2010

Journal of Biological Chemistry

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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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Section: Ar Is Expressed As Two Full-length Receptor Forms-contrasting

confidence: 47%

Section: Methodsmentioning

confidence: 99%

Section: Sds-page Western Blotting and Fluorography-samplesmentioning

confidence: 99%

See 1 more Smart Citation

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

Hakalahti¹,

Vierimaa

Lilja

et al. 2010

Journal of Biological Chemistry

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“…In this report, we describe the general features of CaSR biosynthesis using [ (4,27), vasopressin (10,28), bradykinin (29), and luteinizing hormone (30,31) receptors. [ 35 S]CaSR is stable in the ER form (ϳ140 kDa), and maturation to the endoglycosidase H-resistant form (ϳ160 kDa) is generally observed 16 h after the [ 35 S]cysteine pulse.…”

Section: Discussionmentioning

confidence: 99%

Calcium-sensing Receptor Biosynthesis Includes a Cotranslational Conformational Checkpoint and Endoplasmic Reticulum Retention

Cavanaugh

McKenna

Stepanchick

et al. 2010

Journal of Biological Chemistry

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“…Western blotting as well as fluorography, densitometric scanning, and data analysis for the detection and analysis of radioactively labeled proteins were performed as described previously (18,21). Films were scanned with Umax PowerLook 1120 color scanner and Image Master 2D Platinum 6.0 software.…”

Section: Preparation and Solubilization Of Membranes And Wholementioning

confidence: 99%

N-Glycan-mediated Quality Control in the Endoplasmic Reticulum Is Required for the Expression of Correctly Folded δ-Opioid Receptors at the Cell Surface

Markkanen

Petäjä-Repo

2008

Journal of Biological Chemistry

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A great majority of G protein-coupled receptors are modified by N-glycosylation, but the functional significance of this modification for receptor folding and intracellular transport has remained elusive. Here we studied these phenomena by mutating the two N-terminal N-glycosylation sites (Asn 18 and Asn 33 ) of the human ␦-opioid receptor, and expressing the mutants from the same chromosomal integration site in stably transfected inducible HEK293 cells. Both N-glycosylation sites were used, and their abolishment decreased the steady-state level of receptors at the cell surface. However, pulse-chase labeling, cell surface biotinylation, and immunofluorescence microscopy revealed that this was not because of intracellular accumulation. Instead, the non-N-glycosylated receptors were exported from the endoplasmic reticulum with enhanced kinetics. The results also revealed differences in the significance of the individual N-glycans, as the one attached to Asn 33 was found to be more important for endoplasmic reticulum retention of the receptor. The non-N-glycosylated receptors did not show gross functional impairment, but flow cytometry revealed that a fraction of them was incapable of ligand binding at the cell surface. In addition, the receptors that were devoid of N-glycans showed accelerated turnover and internalization and were targeted for lysosomal degradation. The results accentuate the importance of protein conformation-based screening before export from the endoplasmic reticulum, and demonstrate how the system is compromised when N-glycosylation is disrupted. We conclude that N-glycosylation of the ␦-opioid receptor is needed to maintain the expression of fully functional and stable receptor molecules at the cell surface.

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Inefficient Maturation of the Rat Luteinizing Hormone Receptor

Cited by 73 publications

References 71 publications

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

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

Calcium-sensing Receptor Biosynthesis Includes a Cotranslational Conformational Checkpoint and Endoplasmic Reticulum Retention

N-Glycan-mediated Quality Control in the Endoplasmic Reticulum Is Required for the Expression of Correctly Folded δ-Opioid Receptors at the Cell Surface

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