Effect of N-Glycosylation on Ligand Binding Affinity of Rat V1a Vasopressin Receptor

Lee, Ki Hwan; Ahn, Joon Ik; Yu, Dong; Jeong, Han; Kim, Kyung Soo; Chung, Il Yup; Kim, Jin‐Hyuk; Lee, Yong Sung

doi:10.1006/bbrc.2001.5456

Cited by 7 publications

(8 citation statements)

References 27 publications

(33 reference statements)

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“…2 and 4), allowing the surface expression of ACE2 lacking complex sugars. This finding is consistent with a previous work that showed that inhibition of ER glucosidases with iminosugars did not affect ACE2 expression receptor-ligand binding (69) and N-glycosylation of P2Y12 receptor is necessary to trigger a proper downstream Gi-mediated signaling (70). In contrast, N-glycosylation is not required for the receptor functions of angiotensin II type-1 receptor (71), histamine H2 receptor (72) and the orphan G protein-coupled receptor Gpr176 (68).…”

Section: Sars-cov S Protein But Not the Membrane Fusion Induced By Sars-cov-2 Ssupporting

confidence: 93%

“…There are several examples of the requirement for N-glycosylation in formation of protein-protein interactions. For instance, N-glycosylation of the vasopressin V1a receptor is needed for optimal receptor-ligand binding (69) and N-glycosylation of P2Y12 receptor is necessary to trigger a proper downstream Gi-mediated signaling (70). In contrast, N-glycosylation is not required for the receptor functions of angiotensin II type-1 receptor (71), histamine H2 receptor (72) and the orphan G protein-coupled receptor Gpr176 (68).…”

Section: Discussionmentioning

confidence: 99%

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Analysis of the role of N-linked glycosylation in cell-surface expression, function and binding properties of SARS-CoV-2 receptor ACE2

Brandariz-Núñez

Rowland

2021

Preprint

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Human angiotensin I-converting enzyme 2 (hACE2) is a type-I transmembrane glycoprotein that serves as the major cell entry receptor for SARS-CoV and SARS-CoV-2. The viral spike (S) protein is required for attachment to ACE2 and subsequent virus-host cell membrane fusion. Previous work has demonstrated the presence of N-linked glycans in ACE2. N-glycosylation is implicated in many biological activities, including protein folding, protein activity, and cell surface expression of biomolecules. However, the contribution of N-glycosylation to ACE2 function is poorly understood. Here, we examined the role of N-glycosylation in the activity and localization of two species with different susceptibility to SARS-CoV-2 infection, porcine ACE2 (pACE2) and hACE2. The elimination of N-glycosylation by tunicamycin (TM) treatment or mutagenesis, showed that N-glycosylation is critical for the proper cell surface expression of ACE2 but not for its carboxiprotease activity. Furthermore, nonglycosylable ACE2 localized predominantly in the endoplasmic reticulum (ER) and not at the cell surface. Our data also revealed that binding of SARS-CoV and SARS-CoV-2 S protein to porcine or human ACE2 was not affected by deglycosylation of ACE2 or S proteins, suggesting that N-glycosylation plays no role in the interaction between SARS coronaviruses and the ACE2 receptor. Impairment of hACE2 N-glycosylation decreased cell to cell fusion mediated by SARS-CoV S protein but not SARS-CoV-2 S protein. Finally, we found that hACE2 N-glycosylation is required for an efficient viral entry of SARS-CoV/SARS-CoV-2 S pseudotyped viruses, which could be the result of low cell surface expression of the deglycosylated ACE2 receptor.

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Section: Sars-cov S Protein But Not the Membrane Fusion Induced By Sars-cov-2 Ssupporting

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Analysis of the role of N-linked glycosylation in cell-surface expression, function and binding properties of SARS-CoV-2 receptor ACE2

Brandariz-Núñez

Rowland

2021

Preprint

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“…Importantly, the functional significance of N-glycosylation differs between GPCRs (see ref. [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]. In the case of Gpr176, N-glycosylation is required for its efficient protein expression.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, depending on the type of receptor examined, N-glycosylation can be either critical or non-critical for GPCR function. For example, N-glycosylation of the vasopressin V1a receptor is critical to maintain optimal ligand binding affinity 25 and N-glycosylation of P2Y 12 receptor is required to induce proper downstream G i -mediated signalling 26 . By contrast, N-glycosylation is not needed for the receptor functions of rhodopsin 15 , histamine H 2 receptor 10 , and angiotensin II type-1 receptor 17 .…”

Section: Discussionmentioning

confidence: 99%

Identification and functional characterisation of N-linked glycosylation of the orphan G protein-coupled receptor Gpr176

Wang

Nakagawa

Miyake

et al. 2020

Sci Rep

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G-protein-coupled receptors (GPCRs) are important drug targets with diverse therapeutic applications. However, there are still more than a hundred orphan GPCRs, whose protein functions and biochemical features remain unidentified. Gpr176 encodes a class-A orphan GPCR that has a role in circadian clock regulation in mouse hypothalamus and is also implicated in human breast cancer transcriptional response. Here we show that Gpr176 is N-glycosylated. Peptide-N-glycosidase treatment of mouse hypothalamus extracts revealed that endogenous Gpr176 undergoes N-glycosylation. Using a heterologous expression system, we show that N-glycosylation occurs at four conserved asparagine residues in the N-terminal region of Gpr176. Deficient N-glycosylation due to mutation of these residues reduced the protein expression of Gpr176. At the molecular function level, Gpr176 has constitutive, agonist-independent activity that leads to reduced cAMP synthesis. Although deficient N-glycosylation did not compromise this intrinsic activity, the resultant reduction in protein expression was accompanied by attenuation of cAMP-repressive activity in the cells. We also demonstrate that human GPR176 is N-glycosylated. Importantly, missense variations in the conserved N-glycosylation sites of human GPR176 (rs1473415441; rs761894953) affected N-glycosylation and thereby attenuated protein expression and cAMP-repressive activity in the cells. We show that N-glycosylation is a prerequisite for the efficient protein expression of functional Gpr176/GPR176. G-protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors and are the therapeutic targets of nearly a third of clinically marketed drugs 1,2. Despite their importance, more than one hundred human GPCRs remain poorly characterised due to the lack of useful information on their ligands 3. Included among these so-called orphan GPCRs is GPR176, which is predicted to be a 56-kDa seven-transmembrane protein of class A GPCR with potential sites for N-glycosylation. GPR176 (also known as HB-954) was initially cloned by Hata et al. from a human brain cDNA library 4. In the mouse brain, Gpr176 mRNA levels are predominantly high in the suprachiasmatic nucleus of the hypothalamus (SCN) 5 , the principal circadian pacemaker in mammals, and knockout studies have shown that Gpr176 is required to set the pace of circadian rhythm in behaviour 5. This gene is also expressed in other tissues than the brain 4 and was reported to be involved in the anacardic acid-induced transcriptional response of human breast cancer cells 6. Gpr176 couples to Gz, a subtype of Gi/o, and even in the absence of a known ligand, Gpr176 possesses an agonist-independent constitutive activity that leads to reduced cAMP synthesis 5,7. At the amino acid sequence level, Gpr176 contains five extracellular potential sites for N-glycosylation (Asn-X-Ser/Thr, where X is any amino acid except for Pro); one is located in the third extracellular loop (ECL3) and all other four are located in the N-terminal region. However, wh...

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“…As a result of the lack of glycosylation, GPCRs are mislocated in the cell and, in some cases, this leads to diminished or even abolished coupling, as is the case for the human VPAC 1 vasoactive intestinal peptide receptor, rat AT 1a angiotensin‐II receptor and human nephrin receptor [11–13]. The absence of N‐glycosylation frequently results in a decreased expression level at the cell surface, as occurs for rat EP 3 prostaglandin receptor, human AT 1 angiotensin‐II receptor, human 5‐HT 5A serotonin receptor, human B 2 bradykinin receptor, human TXA 2 thromboxane receptor and human D 5 dopamine receptor [14–19]; however, in some cases, the nonglycosylated receptors are fully functional and properly localized at the plasma membrane, as is the case for rat V 1a vasopressin receptor, canine H 2 histamine receptor, porcine M 2 muscarinic acetylcholine receptor, as well as human D 1 and D 4 dopamine receptors [19–23]. The presence and significance of N‐glycosylation of the 5‐HT 7(a) receptor was investigated at the two putative target asparagine residues (N5 and N66) with a particular focus on receptor localization within the cell and the impact upon ligand binding and activation of AC.…”

Section: Introductionmentioning

confidence: 99%

Biochemical and pharmacological study of N‐linked glycosylation of the human serotonin 5‐HT_7(a) receptor

et al. 2012

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The 5-hydroxytryptamine (5-HT) 7(a) receptor is a G-protein-coupled receptor critically involved in human psychiatric and neurological disorders. In the present study, we evaluate the presence and the functional role of N-glycosylation of the human 5-HT 7 receptor. Western blot analysis of HEK293T cells transiently expressing the 5-HT 7(a) receptor in the presence of tunicamycin gave rise to a band shift, indicating the existence of an N-glycosylated form of the 5-HT 7(a) receptor. To further investigate this, we mutated the two predicted N-glycosylation sites (N5Q and N66Q) and compared the molecular mass of the immunoreactive bands with those of the wild-type receptor, indicating that both asparagines were N-glycosylated. The mutant receptors had the same binding affinity for [ 3 H]5-CT and the same potency and efficacy with regard to 5-HT-induced activation of adenylyl cyclase. However, there was a reduction in maximal ligand binding for the single and double mutants compared to the wild-type receptor. Next, membrane labelling and immunocytochemical studies demonstrated that the N-glycosylation mutants were expressed at the cell surface. We conclude that N-glycosylation is not important for cell surface expression of the 5-HT 7 receptor.

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Effect of N-Glycosylation on Ligand Binding Affinity of Rat V1a Vasopressin Receptor

Cited by 7 publications

References 27 publications

Analysis of the role of N-linked glycosylation in cell-surface expression, function and binding properties of SARS-CoV-2 receptor ACE2

Analysis of the role of N-linked glycosylation in cell-surface expression, function and binding properties of SARS-CoV-2 receptor ACE2

Identification and functional characterisation of N-linked glycosylation of the orphan G protein-coupled receptor Gpr176

Biochemical and pharmacological study of N‐linked glycosylation of the human serotonin 5‐HT_7(a) receptor

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Effect of N-Glycosylation on Ligand Binding Affinity of Rat V1a Vasopressin Receptor

Cited by 7 publications

References 27 publications

Analysis of the role of N-linked glycosylation in cell-surface expression, function and binding properties of SARS-CoV-2 receptor ACE2

Analysis of the role of N-linked glycosylation in cell-surface expression, function and binding properties of SARS-CoV-2 receptor ACE2

Identification and functional characterisation of N-linked glycosylation of the orphan G protein-coupled receptor Gpr176

Biochemical and pharmacological study of N‐linked glycosylation of the human serotonin 5‐HT7(a) receptor

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Biochemical and pharmacological study of N‐linked glycosylation of the human serotonin 5‐HT_7(a) receptor