scholarly journals A Point Mutation in the Human Melanin Concentrating Hormone Receptor 1 Reveals an Important Domain for Cellular Trafficking

2005 ◽  
Vol 19 (10) ◽  
pp. 2579-2590 ◽  
Author(s):  
Jun Fan ◽  
Stephen J. Perry ◽  
Yinghong Gao ◽  
David A. Schwarz ◽  
Richard A. Maki
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Point Mutation ◽  
Hormone Receptor ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Single Mutation ◽  
Wild Type ◽  
Melanin Concentrating Hormone ◽  
Type Receptor

Abstract G protein-coupled receptors (GPCRs) are heptahelical integral membrane proteins that require cell surface expression to elicit their effects. The lack of appropriate expression of GPCRs may be the underlying cause of a number of inherited disorders. There is evidence that newly synthesized GPCRs must attain a specific conformation for their correct trafficking to the cell surface. In this study, we show that a single point mutation in human melanin-concentrating hormone receptor (hMCHR1) at position 255 (T255A), which is located at the junction of intracellular loop 3 and transmembrane domain 6, reduces the hMCHR1 cell surface expression level to 20% of that observed for the wild-type receptor. Most of these mutant receptors are located intracellularly, as opposed to the wild-type receptor, which is located primarily on the cell surface. Immunoprecipitation experiments show that hMCHR1-T255A has reduced glycosylation compared with the wild-type receptor and is associated with the chaperone protein, calnexin, and it colocalizes in the endoplasmic reticulum with KDEL-containing proteins. We also demonstrate that a cell-permeable small molecule antagonist of hMCHR1 can function as a pharmacological chaperone to restore cell surface expression of this and other MCHR1 mutants to wild-type levels. Once rescued, the T255A mutant couples to Gq proteins as efficiently as the wild-type receptor. These data suggest that this single mutation produces an hMCHR1 that folds incorrectly, resulting in its retention in the endoplasmic reticulum, but once rescued to the cell surface can still function normally.

The immunogenicity of VP7, a rotavirus antigen resident in the endoplasmic reticulum, is enhanced by cell surface expression.

1990 ◽  
Vol 64 (10) ◽  
pp. 4776-4783 ◽  
Author(s):  
M E Andrew ◽  
D B Boyle ◽  
P L Whitfeld ◽  
L J Lockett ◽  
I D Anthony ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression

scholarly journals Blocking intracellular degradation of the erythropoietin and asialoglycoprotein receptors by calpain inhibitors does not result in the same increase in the levels of their membrane and secreted forms

1996 ◽  
Vol 313 (2) ◽  
pp. 391-399 ◽  
Author(s):  
Drorit NEUMANN ◽  
Ming YUK HUAM ◽  
Harvey F. LODISH ◽  
Gerardo Z. LEDERKREMER
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Membrane Glycoprotein ◽  
3T3 Cells ◽  
Proteolytic Fragment ◽  
Intracellular Degradation ◽  
Nih 3T3 ◽  
Calpain Inhibitors

The erythropoietin receptor (EPO-R), a type 1 membrane glycoprotein, is degraded mainly in the lysosomes or endosomes, whereas the asialoglycoprotein receptor (ASGP-R) H2a subunit, a type 2 membrane glycoprotein, is degraded exclusively in the endoplasmic reticulum. The present study describes compounds that inhibit the intracellular degradation of these receptors in an efficient manner. However, the levels of cell-surface expression and secretion of their soluble exoplasmic domains were not enhanced to the same extent. The calpain inhibitors N-acetyl-leucyl-leucyl-norleucinal(ALLN) and N-acetyl-leucyl-leucyl-methional (ALLM) inhibited EPO-R degradation profoundly. After 3 h of chase using Ba/F3 cells and NIH 3T3 fibroblasts expressing the EPO-R, virtually all of the receptor molecules were degraded, whereas 80% of the pulse-labelled receptor remained intact in the presence of the inhibitor. EPO-R cell-surface expression was elevated 1.5-fold after 1 h of incubation with ALLN. In the absence of protein synthesis, ALLN caused the accumulation of non-degraded EPO-R molecules in endosomes and lysosomes, as determined by double immunofluorescence labelling of NIH 3T3 cells expressing EPO-Rs. In Ba/F3 cells expressing a soluble EPO-R, ALLN treatment increased secretion of the soluble exoplasmic domain of the EPO-R 2-5-fold. Similarly, in NIH 3T3 cells singly transfected with the ASGP-R H2a subunit cDNA, ALLN inhibited degradation of the ASGP-R H2a subunit precursor, as well as the degradation of the 35 kDa proteolytic fragment corresponding to the receptor ectodomain, by 3-6-fold. However, accumulation of secreted proteolytic fragment in the medium was augmented in the presence of ALLN by only 1.75-fold. In cells expressing the G78R mutant of the ASGP-R H2a subunit, which is not cleaved to the 35 kDa fragment [Yuk and Lodish (1993) J. Cell Biol. 123,1735-1749], degradation of the precursor was inhibited. Overall, our data suggest the involvement of cysteine proteinases located in the endoplasmic reticulum, as well as in post-Golgi compartments, in degradation of the EPO-R and the ASGP-R H2a subunit. The much lower effect of the inhibitory compounds on cell-surface and secreted forms of the EPO-R and ASGP-R H2a subunit illustrates the complexity and the tight regulation of the cellular localization and stability of membrane proteins.

scholarly journals Modulation of Transporter Associated with Antigen Processing (TAP)-Mediated Peptide Import into the Endoplasmic Reticulum by Flavivirus Infection

2001 ◽  
Vol 75 (12) ◽  
pp. 5663-5671 ◽  
Author(s):  
Frank Momburg ◽  
Arno Müllbacher ◽  
Mario Lobigs
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Mhc Class I ◽  
Antigen Processing ◽  
Surface Expression ◽  
Class I ◽  
Peptide Transport ◽  
Cell Surface Expression ◽  
Flavivirus Infection ◽  
Mhc Class I Molecules

ABSTRACT In contrast to many other viruses that escape the cellular immune response by downregulating major histocompatibility complex (MHC) class I molecules, flavivirus infection can upregulate their cell surface expression. Previously we have presented evidence that during flavivirus infection, peptide supply to the endoplasmic reticulum is increased (A. Müllbacher and M. Lobigs, Immunity 3:207–214, 1995). Here we show that during the early phase of infection with different flaviviruses, the transport activity of the peptide transporter associated with antigen processing (TAP) is augmented by up to 50%. TAP expression is unaltered during infection, and viral but not host macromolecular synthesis is required for enhanced peptide transport. This study is the first demonstration of transient enhancement of TAP-dependent peptide import into the lumen of the endoplasmic reticulum as a consequence of a viral infection. We suggest that the increased supply of peptides for assembly with MHC class I molecules in flavivirus-infected cells accounts for the upregulation of MHC class I cell surface expression with the biological consequence of viral evasion of natural killer cell recognition.

scholarly journals Serine 129 phosphorylation of membrane-associated α-synuclein modulates dopamine transporter function in a G protein–coupled receptor kinase–dependent manner

2013 ◽  
Vol 24 (11) ◽  
pp. 1649-1660 ◽  
Author(s):  
Susumu Hara ◽  
Shigeki Arawaka ◽  
Hiroyasu Sato ◽  
Youhei Machiya ◽  
Can Cui ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Dopamine Transporter ◽  
Surface Expression ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
Receptor Kinase ◽  
Wild Type ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

Most α-synuclein (α-syn) deposited in Lewy bodies, the pathological hallmark of Parkinson disease (PD), is phosphorylated at Ser-129. However, the physiological and pathological roles of this modification are unclear. Here we investigate the effects of Ser-129 phosphorylation on dopamine (DA) uptake in dopaminergic SH-SY5Y cells expressing α-syn. Subcellular fractionation of small interfering RNA (siRNA)–treated cells shows that G protein–coupled receptor kinase 3 (GRK3), GRK5, GRK6, and casein kinase 2 (CK2) contribute to Ser-129 phosphorylation of membrane-associated α-syn, whereas cytosolic α-syn is phosphorylated exclusively by CK2. Expression of wild-type α-syn increases DA uptake, and this effect is diminished by introducing the S129A mutation into α-syn. However, wild-type and S129A α-syn equally increase the cell surface expression of dopamine transporter (DAT) in SH-SY5Y cells and nonneuronal HEK293 cells. In addition, siRNA-mediated knockdown of GRK5 or GRK6 significantly attenuates DA uptake without altering DAT cell surface expression, whereas knockdown of CK2 has no effect on uptake. Taken together, our results demonstrate that membrane-associated α-syn enhances DA uptake capacity of DAT by GRKs-mediated Ser-129 phosphorylation, suggesting that α-syn modulates intracellular DA levels with no functional redundancy in Ser-129 phosphorylation between GRKs and CK2.

scholarly journals Misfolding Ectodomain Mutations of the Lutropin Receptor Increase Efficacy of Hormone Stimulation

2016 ◽  
Vol 30 (1) ◽  
pp. 62-76 ◽  
Author(s):  
E. Charmandari ◽  
R. Guan ◽  
M. Zhang ◽  
L. G. Silveira ◽  
Q. R. Fan ◽  
...  
Keyword(s):  
Cell Surface ◽  
Leydig Cells ◽  
Cellular Response ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Lutropin Receptor ◽  
Mutant Receptors ◽  
Type Receptor ◽  
Leydig Cell Hypoplasia ◽  
Inactivating Mutations

Abstract We demonstrate 2 novel mutations of the LHCGR, each homozygous, in a 46,XY patient with severe Leydig cell hypoplasia. One is a mutation in the signal peptide (p.Gln18_Leu19ins9; referred to here as SP) that results in an alteration of the coding sequence of the N terminus of the mature mutant receptor. The other mutation (p.G71R) is also within the ectodomain. Similar to many other inactivating mutations, the cell surface expression of recombinant human LHR(SP,G71R) is greatly reduced due to intracellular retention. However, we made the unusual discovery that the intrinsic efficacy for agonist-stimulated cAMP in the reduced numbers of receptors on the cell surface was greatly increased relative to the same low number of cell surface wild-type receptor. Remarkably, this appears to be a general attribute of misfolding mutations in the ectodomains, but not serpentine domains, of the gonadotropin receptors. These findings suggest that there must be a common, shared mechanism by which disparate mutations in the ectodomain that cause misfolding and therefore reduced cell surface expression concomitantly confer increased agonist efficacy to those receptor mutants on the cell surface. Our data further suggest that, due to their increased agonist efficacy, extremely small changes in cell surface expression of misfolded ectodomain mutants cause larger than expected alterations in the cellular response to agonist. Therefore, for inactivating LHCGR mutations causing ectodomain misfolding, the numbers of cell surface mutant receptors on fetal Leydig cells of 46,XY individuals exert a more exquisite effect on the relative severity of the clinical phenotypes than already appreciated.

scholarly journals A Highly Conserved Motif at the COOH Terminus Dictates Endoplasmic Reticulum Exit and Cell Surface Expression of NKCC2

2009 ◽  
Vol 284 (32) ◽  
pp. 21752-21764 ◽  
Author(s):  
Nancy Zaarour ◽  
Sylvie Demaretz ◽  
Nadia Defontaine ◽  
David Mordasini ◽  
Kamel Laghmani
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Conserved Motif

scholarly journals Cell Surface Expression of Calnexin, a Molecular Chaperone in the Endoplasmic Reticulum

2000 ◽  
Vol 275 (46) ◽  
pp. 35751-35758 ◽  
Author(s):  
Yasushi Okazaki ◽  
Hiroshi Ohno ◽  
Kan Takase ◽  
Takenori Ochiai ◽  
Takashi Saito
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Molecular Chaperone ◽  
Surface Expression ◽  
Cell Surface Expression
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