scholarly journals The Role of Cysteines and Charged Amino Acids in Extracellular Loops of the Human Ca2+Receptor in Cell Surface Expression and Receptor Activation Processes

Endocrinology ◽  
2004 ◽  
Vol 145 (8) ◽  
pp. 3892-3903 ◽  
Author(s):  
Kausik Ray ◽  
Sanchita P. Ghosh ◽  
John K. Northup
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Surface Expression ◽  
Receptor Activation ◽  
Cell Surface Expression ◽  
Charged Amino Acids ◽  
Extracellular Loops

The role of the extracellular loops of the CGRP receptor, a family B GPCR

2012 ◽  
Vol 40 (2) ◽  
pp. 433-437 ◽  
Author(s):  
James Barwell ◽  
Michael J. Woolley ◽  
Mark Wheatley ◽  
Alex C. Conner ◽  
David R. Poyner
Keyword(s):  
Surface Expression ◽  
Receptor Activation ◽  
Receptor Expression ◽  
Cell Surface Expression ◽  
Calcitonin Receptor ◽  
Transmembrane Helices ◽  
Extracellular Loops ◽  
Calcitonin Gene ◽  
G Protein Coupled

The CGRP (calcitonin gene-related peptide) receptor is a family B GPCR (G-protein-coupled receptor). It consists of a GPCR, CLR (calcitonin receptor-like receptor) and an accessory protein, RAMP1 (receptor activity-modifying protein 1). RAMP1 is needed for CGRP binding and also cell-surface expression of CLR. There have been few systematic studies of the ECLs (extracellular loops) of family B GPCRs. However, they are likely to be especially important for the interaction of the N-termini of the peptide agonists that are the natural agonists for these receptors. We have carried out alanine scans on all three ECLs of CLR, as well as their associated juxtamembrane regions. Residues within all three loops influence CGRP binding and receptor activation. Mutation of Ala203 and Ala206 on ECL1 to leucine increased the affinity of CGRP. Residues at the top of TM (transmembrane) helices 2 and 3 influenced CGRP binding and receptor activation. L351A and E357A in TM6/ECL3 reduced receptor expression and may be needed for CLR association with RAMP1. ECL2 seems especially important for CLR function; of the 16 residues so far examined in this loop, eight residues reduce the potency of CGRP at stimulating cAMP production when mutated to alanine.

scholarly journals Variant Amino Acids in the Extracellular Loops of Murine and Human Vasopressin V2 Receptors Account for Differences in Cell Surface Expression and Ligand Affinity

2002 ◽  
Vol 16 (4) ◽  
pp. 799-813 ◽  
Author(s):  
Alexander Oksche ◽  
Gabriele Leder ◽  
Susanne Valet ◽  
Matthias Platzer ◽  
Kerstin Hasse ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Ligand Affinity ◽  
Extracellular Loops

scholarly journals Comparative Mutagenesis of Pseudorabies Virus and Epstein-Barr Virus gH Identifies a Structural Determinant within Domain III of gH Required for Surface Expression and Entry Function

2015 ◽  
Vol 90 (5) ◽  
pp. 2285-2293 ◽  
Author(s):  
Britta S. Möhl ◽  
Christina Schröter ◽  
Barbara G. Klupp ◽  
Walter Fuchs ◽  
Thomas C. Mettenleiter ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
B Cell ◽  
Epstein Barr Virus ◽  
Pseudorabies Virus ◽  
Surface Expression ◽  
Structural Motif ◽  
Cell Surface Expression ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACTHerpesviruses infect cells using the conserved core fusion machinery composed of glycoprotein B (gB) and gH/gL. The gH/gL complex plays an essential but still poorly characterized role in membrane fusion and cell tropism. Our previous studies demonstrated that the conserved disulfide bond (DB) C278/C335 in domain II (D-II) of Epstein-Barr virus (EBV) gH has an epithelial cell-specific function, whereas the interface of D-II/D-III is involved in formation of the B cell entry complex by binding to gp42. To extend these studies, we compared gH of the alphaherpesvirus pseudorabies virus (PrV) with gH of the gammaherpesvirus EBV to identify functionally equivalent regions critical for gH function during entry. We identified several conserved amino acids surrounding the conserved DB that connects three central helices of D-III of PrV and EBV gH. The present study verified that the conserved DB and several contacting amino acids in D-III modulate cell surface expression and thereby contribute to gH function. In line with this finding, we found that DB C404/C439 and T401 are important for cell-to-cell spread and efficient entry of PrV. This parallel comparison between PrV and EBV gH function brings new insights into how gH structure impacts fusion function during herpesvirus entry.IMPORTANCEThe alphaherpesvirus PrV is known for its neuroinvasion, whereas the gammaherpesvirus EBV is associated with cancer of epithelial and B cell origin. Despite low amino acid conservation, PrV gH and EBV gH show strikingly similar structures. Interestingly, both PrV gH and EBV gH contain a structural motif composed of a DB and supporting amino acids which is highly conserved within theHerpesviridae. Our study verified that PrV gH uses a minimal motif with the DB as the core, whereas the DB of EBV gH forms extensive connections through hydrogen bonds to surrounding amino acids, ensuring the cell surface expression of gH/gL. Our study verifies that the comparative analysis of distantly related herpesviruses, such as PrV and EBV, allows the identification of common gH functions. In addition, we provide an understanding of how functional domains can evolve over time, resulting in subtle differences in domain structure and function.

scholarly journals Analysis of the Role of N-Linked Glycosylation in Cell Surface Expression, Function, and Binding Properties of SARS-CoV-2 Receptor ACE2

2021 ◽  
Author(s):  
Raymond Rowland ◽  
Alberto Brandariz-Nuñez
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Receptor Interaction ◽  
Minimal Effect ◽  
Binding Properties ◽  
Virus Receptor

Understanding the role of glycosylation in the virus-receptor interaction is important for developing approaches that disrupt infection. In this study, we showed that deglycosylation of both ACE2 and S had a minimal effect on the spike-ACE2 interaction.

scholarly journals C-terminal region regulates the functional expression of human noradrenaline transporter splice variants

2006 ◽  
Vol 401 (1) ◽  
pp. 185-195 ◽  
Author(s):  
Chiharu Sogawa ◽  
Kei Kumagai ◽  
Norio Sogawa ◽  
Katsuya Morita ◽  
Toshihiro Dohi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Splice Variants ◽  
Functional Expression ◽  
Surface Expression ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
C Terminus ◽  
And Function

The NET [noradrenaline (norepinephrine) transporter], an Na+/Cl−-dependent neurotransmitter transporter, has several isoforms produced by alternative splicing in the C-terminal region, each differing in expression and function. We characterized the two major isoforms of human NET, hNET1, which has seven C-terminal amino acids encoded by exon 15, and hNET2, which has 18 amino acids encoded by exon 16, by site-directed mutagenesis in combination with NE (noradrenaline) uptake assays and cell surface biotinylation. Mutants lacking one third or more of the 24 amino acids encoded by exon 14 exhibited neither cell surface expression nor NE uptake activity, with the exception of the mutant lacking the last eight amino acids of hNET2, whose expression and uptake resembled that of the WT (wild-type). A triple alanine replacement of a candidate motif (ENE) in this region mimicked the influences of the truncation. Deletion of either the last three or another four amino acids of the C-terminus encoded by exon 15 in hNET1 reduced the cell surface expression and NE uptake, whereas deletion of all seven residues reduced the transport activity but did not affect the cell surface expression. Replacement of RRR, an endoplasmic reticulum retention motif, by alanine residues in the C-terminus of hNET2 resulted in a similar expression and function compared with the WT, while partly recovering the effects of the mutation of ENE. These findings suggest that in addition to the function of the C-terminus, the common proximal region encoded by exon 14 regulates the functional expression of splice variants, such as hNET1 and hNET2.

scholarly journals Functional Analysis of the Murine Cytomegalovirus Chemokine Receptor Homologue M33: Ablation of Constitutive Signaling Is Associated with an Attenuated Phenotype In Vivo

2007 ◽  
Vol 82 (4) ◽  
pp. 1884-1898 ◽  
Author(s):  
Ruth Case ◽  
Emma Sharp ◽  
Tau Benned-Jensen ◽  
Mette M. Rosenkilde ◽  
Nicholas Davis-Poynter ◽  
...  
Keyword(s):  
Cell Surface ◽  
Salivary Glands ◽  
Surface Expression ◽  
Receptor Activation ◽  
Murine Cytomegalovirus ◽  
Cell Surface Expression ◽  
Transmembrane Receptors ◽  
C Terminus ◽  
The Impact

ABSTRACT The murine cytomegalovirus (MCMV) M33 gene is conserved among all betaherpesviruses and encodes a homologue of seven-transmembrane receptors (7TMR) with the capacity for constitutive signaling. Previous studies have demonstrated that M33 is important for MCMV dissemination to or replication within the salivary glands. In this study, we probed N- and C-terminal regions of M33 as well as known 7TMR signature motifs in transmembrane (TM) II and TM III to determine the impact on cell surface expression, constitutive signaling, and in vivo phenotype. The region between amino acids R340 and A353 of the C terminus was found to be important for CREB- and NFAT-mediated signaling, although not essential for phosphatidylinositol turnover. Tagging or truncation of the N terminus of M33 resulted in loss of cell surface expression. Within TM II, an F79D mutation abolished constitutive signaling, demonstrating a role, as in other cellular and viral 7TMR, of TM II in receptor activation. In TM III, the arginine (but not the asparagine) residue of the NRY motif (the counterpart of the common DRY motif in cellular 7TMR) was found to be essential for constitutive signaling. Selected mutations incorporated into recombinant MCMV showed that disruption of constitutive signaling for a viral 7TMR homologue resulted in a reduced capacity to disseminate to or replicate in the salivary glands. In addition, HCMV UL33 was found to partially compensate for the lack of M33 in vivo, suggesting conserved biological roles of the UL33 gene family.

Identification of amino acids in the pore region of Kv1.2 potassium channel that regulate its glycosylation and cell surface expression

2010 ◽  
Vol 112 (4) ◽  
pp. 913-923 ◽  
Author(s):  
Iku Utsunomiya ◽  
Shinya Tanabe ◽  
Tomonori Terashi ◽  
Souichi Ikeno ◽  
Tadashi Miyatake ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Potassium Channel ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Pore Region
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