scholarly journals Individual cells traffic the Vasopressin 2 Receptor to their cell surface with different success

2021 ◽  
Author(s):  
Eline J Koers ◽  
Bradley A Morgan ◽  
Iain B Styles ◽  
Dmitry J Veprintsev
Keyword(s):  
Cell Surface ◽  
Receptor Cell ◽  
Surface Expression ◽  
G Protein Coupled Receptors ◽  
Cell Surface Expression ◽  
Subcellular Localisation ◽  
Equal Distribution ◽  
Mutant Receptors ◽  
G Protein Coupled ◽  
Correct Expression

G protein coupled receptors (GPCRs) translate the actions of hormones and neurotransmitters into intracellular signalling events. Mutations in GPCRs can prevent their correct expression and trafficking to the cell surface and cause disease. Single cell subcellular localisation measurements reveal that while some cells appear to traffic the majority of the vasopressin 2 receptor (V2R) molecules to the cell surface, others retain a greater number of receptors in the ER or have approximately equal distribution. Mutations in the V2R affect the proportion of cells able to send this GPCR to their cell surface but surprisingly they do not prevent all cells from correctly trafficking the mutant receptors. These findings reveal the potential for rescue of mutant receptor cell surface expression by pharmacological manipulation of the GPCR folding and trafficking machinery.

scholarly journals Structural instability and divergence from conserved residues underlie intracellular retention of mammalian odorant receptors

2020 ◽  
Vol 117 (6) ◽  
pp. 2957-2967
Author(s):  
Kentaro Ikegami ◽  
Claire A. de March ◽  
Maira H. Nagai ◽  
Soumadwip Ghosh ◽  
Matthew Do ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Surface Expression ◽  
Structural Instability ◽  
G Protein Coupled Receptors ◽  
Machine Learning Techniques ◽  
Odorant Receptors ◽  
Cell Surface Expression ◽  
Conserved Residues ◽  
G Protein Coupled

Mammalian odorant receptors are a diverse and rapidly evolving set of G protein-coupled receptors expressed in olfactory cilia membranes. Most odorant receptors show little to no cell surface expression in nonolfactory cells due to endoplasmic reticulum retention, which has slowed down biochemical studies. Here we provide evidence that structural instability and divergence from conserved residues of individual odorant receptors underlie intracellular retention using a combination of large-scale screening of odorant receptors cell surface expression in heterologous cells, point mutations, structural modeling, and machine learning techniques. We demonstrate the importance of conserved residues by synthesizing consensus odorant receptors that show high levels of cell surface expression similar to conventional G protein-coupled receptors. Furthermore, we associate in silico structural instability with poor cell surface expression using molecular dynamics simulations. We propose an enhanced evolutionary capacitance of olfactory sensory neurons that enable the functional expression of odorant receptors with cryptic mutations.

scholarly journals Structural instability and divergence from conserved residues underlie intracellular retention of mammalian odorant receptors

2019 ◽  
Author(s):  
Kentaro Ikegami ◽  
Claire A. de March ◽  
Maira H. Nagai ◽  
Soumadwip Ghosh ◽  
Matthew Do ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Surface Expression ◽  
Structural Instability ◽  
G Protein Coupled Receptors ◽  
Odorant Receptors ◽  
Cell Surface Expression ◽  
Conserved Residues ◽  
Olfactory Cells ◽  
G Protein Coupled

AbstractMammalian odorant receptors are a diverse and rapidly evolving set of G protein-coupled receptors expressed in olfactory cilia membranes. Most odorant receptors show little to no cell surface expression in non-olfactory cells due to endoplasmic reticulum retention, which has slowed down biochemical studies. Here, we provide evidence that structural instability and divergence from conserved residues of individual odorant receptors underlie intracellular retention using a combination of large-scale screening of odorant receptors cell surface expression in heterologous cells, point mutations, structural modeling, and machine learning techniques. We demonstrate the importance of conserved residues by synthesizing “consensus” odorant receptors that show high levels of cell surface expression similar to conventional G protein-coupled receptors. Furthermore, we associate in silico structural instability with poor cell surface expression using molecular dynamics simulations. We propose an enhanced evolutionary capacitance of olfactory sensory neurons that enable the functional expression of odorant receptors with cryptic mutations.Significance StatementOdor detection in mammals depends on the largest family of G protein-coupled receptors, the odorant receptors, which represent ∼2% of our protein-coding genes. The vast majority of odorant receptors are trapped within the cell when expressed in non-olfactory cells. The underlying causes of why odorant receptors cannot be functionally expressed in non-olfactory cells have remained enigmatic for over 20 years. Our study points to divergence from a consensus sequence as a key factor in a receptor’s inability to function in non-olfactory cells, which in turn, helps explain odorant receptors’ exceptional functional diversity and rapid evolution. We also show the success of protein engineering strategies for promoting odorant receptor cell surface expression.

scholarly journals Genetic Variations in the Human G Protein-coupled Receptor Class C, Group 6, Member A (GPRC6A) Control Cell Surface Expression and Function

2016 ◽  
Vol 292 (4) ◽  
pp. 1524-1534 ◽  
Author(s):  
Stine Jørgensen ◽  
Christian Theil Have ◽  
Christina Rye Underwood ◽  
Lars Dan Johansen ◽  
Petrine Wellendorph ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Control Cell ◽  
Surface Expression ◽  
Genetic Variations ◽  
Cell Surface Expression ◽  
G Protein Coupled Receptor ◽  
Group 6 ◽  
And Function ◽  
G Protein Coupled

scholarly journals Assembly-dependent Surface Targeting of the Heterodimeric GABAB Receptor Is Controlled by COPI but Not 14-3-3

2005 ◽  
Vol 16 (12) ◽  
pp. 5572-5578 ◽  
Author(s):  
Carsten Brock ◽  
Laure Boudier ◽  
Damien Maurel ◽  
Jaroslav Blahos ◽  
Jean-Philippe Pin
Keyword(s):  
Quality Control ◽  
Cell Surface ◽  
Control Mechanism ◽  
Gabab Receptor ◽  
Transmembrane Proteins ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Proper Function ◽  
G Protein Coupled ◽  
Retention Signal

Cell surface expression of transmembrane proteins is strictly regulated. Mutually exclusive interaction with COPI or 14-3-3 proteins has been proposed as a mechanism underlying such trafficking control of various proteins. In particular, 14-3-3 dimers have been proposed to “sense” correctly assembled oligomers, allowing their surface targeting by preventing COPI-mediated intracellular retention. Here we examined whether such a mechanism is involved in the quality control of the heterodimeric G protein-coupled GABAB receptor. Its GB1 subunit, carrying the retention signal RSR, only reaches the cell surface when associated with the GB2 subunit. We show that COPI and 14-3-3 specifically bind to the GB1 RSR sequence and that COPI is involved in its intracellular retention. However, we demonstrate that the interaction with 14-3-3 is not required for proper function of the GABAB receptor quality control. Accordingly, competition between 14-3-3 and COPI cannot be considered as a general trafficking control mechanism. A possible other role for competition between COPI and 14-3-3 binding is discussed.

scholarly journals GGA3 Interacts with a G Protein-Coupled Receptor and Modulates Its Cell Surface Export

2016 ◽  
Vol 36 (7) ◽  
pp. 1152-1163 ◽  
Author(s):  
Maoxiang Zhang ◽  
Jason E. Davis ◽  
Chunman Li ◽  
Jie Gao ◽  
Wei Huang ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Molecular Mechanisms ◽  
Specific Interaction ◽  
Adaptor Protein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Intracellular Loop ◽  
Interaction Sites ◽  
G Protein Coupled

Molecular mechanisms governing the anterograde trafficking of nascent G protein-coupled receptors (GPCRs) are poorly understood. Here, we have studied the regulation of cell surface transport of α2-adrenergic receptors (α2-ARs) by GGA3 (Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding protein 3), a multidomain clathrin adaptor protein that sorts cargo proteins at thetrans-Golgi network (TGN) to the endosome/lysosome pathway. By using an inducible system, we demonstrated that GGA3 knockdown significantly inhibited the cell surface expression of newly synthesized α2B-AR without altering overall receptor synthesis and internalization. The receptors were arrested in the TGN. Furthermore, GGA3 knockdown attenuated α2B-AR-mediated signaling, including extracellular signal-regulated kinase 1/2 (ERK1/2) activation and cyclic AMP (cAMP) inhibition. More interestingly, GGA3 physically interacted with α2B-AR, and the interaction sites were identified as the triple Arg motif in the third intracellular loop of the receptor and the acidic motif EDWE in the VHS domain of GGA3. In contrast, α2A-AR did not interact with GGA3 and its cell surface export and signaling were not affected by GGA3 knockdown. These data reveal a novel function of GGA3 in export trafficking of a GPCR that is mediated via a specific interaction with the receptor.

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 Regulation of GIP and GLP1 Receptor Cell Surface Expression by N-Glycosylation and Receptor Heteromerization

PLoS ONE ◽  
2012 ◽  
Vol 7 (3) ◽  
pp. e32675 ◽  
Author(s):  
Gina M. Whitaker ◽  
Francis C. Lynn ◽  
Christopher H. S. McIntosh ◽  
Eric A. Accili
Keyword(s):  
Cell Surface ◽  
Receptor Cell ◽  
Surface Expression ◽  
Cell Surface Expression

scholarly journals RACK1 Regulates the Cell Surface Expression of the G Protein-Coupled Receptor for Thromboxane A2

Traffic ◽  
2008 ◽  
Vol 9 (3) ◽  
pp. 394-407 ◽  
Author(s):  
Audrey Parent ◽  
Geneviève Laroche ◽  
Émilie Hamelin ◽  
Jean-Luc Parent
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Thromboxane A2 ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

scholarly journals The Low-Density Lipoprotein Class A Module of the Relaxin Receptor (Leucine-Rich Repeat Containing G-Protein Coupled Receptor 7): Its Role in Signaling and Trafficking to the Cell Membrane

Endocrinology ◽  
2007 ◽  
Vol 148 (3) ◽  
pp. 1181-1194 ◽  
Author(s):  
András Kern ◽  
Alexander I. Agoulnik ◽  
Gillian D. Bryant-Greenwood
Keyword(s):  
Cell Surface ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Lipoprotein Class ◽  
Class A ◽  
Relaxin Family ◽  
G Protein Coupled ◽  
Relaxin Receptor

The relaxin receptor (LGR7, relaxin family peptide receptor 1) is a member of the leucine-rich repeat containing G protein-coupled receptors subgroup C. This and the LGR8 (relaxin family peptide receptor 2) receptor are unique in having a low-density lipoprotein class A (LDL-A) module at their N termini. This study was designed to show the role of the LDL-A in LGR7 expression and function. Point mutants for the conserved cysteines (Cys47 and Cys53) and for calcium binding asparagine (Asp58), a mutant with deleted LDL-A domain and chimeric LGR7 receptor with LGR8 LDL-A all showed no cAMP response to human relaxins H1 or H2. We have shown that their cell surface delivery was uncompromised. The mutation of the putative N-linked glycosylation site (Asn36) decreased cAMP production and reduced cell surface expression to 37% of the wild-type LGR7. All point mutant, chimeric, and wild-type receptor proteins were expressed as the two forms. The immature or precursor form of the receptor was 80 kDa, whereas the mature receptor, delivered to the cell surface was 95 kDa. The glycosylation mutant was also expressed as two forms with appropriately smaller molecular masses. Deletion of the LDL-A module resulted in expression of the mature receptor only. These data suggest that the LDL-A module of LGR7 influences receptor maturation, cell surface expression, and relaxin-activated signal transduction.

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