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 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 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 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 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 Mutational analysis of the R33-encoded G protein-coupled receptor of rat cytomegalovirus: identification of amino acid residues critical for cellular localization and ligand-independent signalling

2004 ◽  
Vol 85 (4) ◽  
pp. 897-909 ◽  
Author(s):  
Yvonne K. Gruijthuijsen ◽  
Erik V. H. Beuken ◽  
Martine J. Smit ◽  
Rob Leurs ◽  
Cathrien A. Bruggeman ◽  
...  
Keyword(s):  
G Protein ◽  
Cellular Localization ◽  
Mutational Analysis ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Constitutive Activity ◽  
G Protein Coupled Receptor ◽  
Human Cmv ◽  
G Protein Coupled

The rat cytomegalovirus (RCMV) R33 gene encodes a G protein-coupled receptor (GPCR), pR33, which possesses agonist-independent, constitutive signalling activity. To characterize this activity further, we generated a series of point and deletion mutants of pR33. Both expression of and signalling by the mutants was evaluated. Several point mutants were generated that contained modifications in the NRY motif. This motif, at aa 130–132 of pR33, is the counterpart of the common DRY motif of GPCRs, which is known to be involved in G protein coupling. We found that mutation of the asparagine residue within the NRY motif of pR33 (N130) to aspartic acid resulted in a mutant (N130D) with similar signalling characteristics to the wild-type (WT) protein, indicating that N130 is not the determinant of constitutive activity of pR33. Interestingly, a mutant carrying an alanine at aa 130 (N130A) was severely impaired in Gq/11-mediated, constitutive activation of phospholipase C, whereas it displayed similar levels of activity to pR33 in Gi/0-mediated signalling. Another protein that contained a modified NRY motif, R131A, did not show constitutive activity, whereas mutants Y132F and Y132A displayed similar activities to the WT receptor. This indicated that residue R131 is critical for pR33 function in vitro, whereas Y132 is not. Finally, we identified two consecutive arginines within the C-terminal tails of both pR33 and its homologue from human CMV, pUL33, which are important for correct cell-surface expression of these receptors.

scholarly journals A G protein–coupled receptor and the intracellular synthase of its agonist functionally cooperate

2014 ◽  
Vol 204 (3) ◽  
pp. 377-393 ◽  
Author(s):  
Chantal Binda ◽  
Samuel Génier ◽  
Andréane Cartier ◽  
Jean-François Larrivée ◽  
Jana Stankova ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Prostaglandin D2 ◽  
Autocrine Signaling ◽  
Independent Manner ◽  
Signaling Mechanism ◽  
Dependent Activity ◽  
G Protein Coupled

Export of newly synthesized G protein–coupled receptors (GPCRs) remains poorly characterized. We show in this paper that lipocalin-type prostaglandin D2 (PGD2) synthase (L-PGDS) interacts intracellularly with the GPCR DP1 in an agonist-independent manner. L-PGDS promotes cell surface expression of DP1, but not of other GPCRs, in HEK293 and HeLa cells, independent of L-PGDS enzyme activity. In addition, formation of a DP1–Hsp90 complex necessary for DP1 export to the cell surface is dependent on the interaction between L-PGDS and the C-terminal MEEVD residues of Hsp90. Surprisingly, PGD2 synthesis by L-PGDS is promoted by coexpression of DP1, suggesting a possible intracrine/autocrine signaling mechanism. In this regard, L-PGDS increases the formation of a DP1–ERK1/2 complex and increases DP1-mediated ERK1/2 signaling. Our findings define a novel cooperative mechanism in which a GPCR (DP1) promotes the activity of the enzyme (L-PGDS) that produces its agonist (PGD2) and in which this enzyme in turn acts as a cofactor (of Hsp90) to promote export and agonist-dependent activity of the receptor.

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.

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