Cell Culture Bioreactor Optimization: Growing Recombinant HEK293 Cells to Produce a G-Protein-Coupled Receptor (rGPCR) Membrane Protein

2010 ◽  
Vol 8 (4) ◽  
pp. 47-50
Author(s):  
Christopher Min ◽  
Yan Jin ◽  
Vikram Gossain
Keyword(s):  
Cell Culture ◽  
Membrane Protein ◽  
G Protein ◽  
Hek293 Cells ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

scholarly journals Vitamin E-based glycoside amphiphiles for membrane protein structural studies

2018 ◽  
Vol 16 (14) ◽  
pp. 2489-2498 ◽  
Author(s):  
Muhammad Ehsan ◽  
Yang Du ◽  
Iago Molist ◽  
Alpay B. Seven ◽  
Parameswaran Hariharan ◽  
...  
Keyword(s):  
Vitamin E ◽  
Membrane Protein ◽  
G Protein ◽  
Structural Studies ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled ◽  
Novel Agent

A vitamin E-based novel agent (i.e., VEG-3) was markedly effective at stabilizing and visualizing a G-protein coupled receptor (GPCR)-Gs complex.

A generic microfluidic biosensor of G protein-coupled receptor activation – impedance measurements of reversible morphological changes of reverse transfected HEK293 cells on microelectrodes

RSC Advances ◽  
2015 ◽  
Vol 5 (65) ◽  
pp. 52563-52570 ◽  
Author(s):  
Saurabh K. Srivastava ◽  
Rajesh Ramaneti ◽  
Margriet Roelse ◽  
Hien Duy Tong ◽  
Elwin X. Vrouwe ◽  
...  
Keyword(s):  
G Protein ◽  
Morphological Changes ◽  
Receptor Activation ◽  
Membrane Receptors ◽  
Hek293 Cells ◽  
G Protein Coupled Receptor ◽  
Impedance Measurements ◽  
Transfected Cells ◽  
Microfluidic Biosensor ◽  
G Protein Coupled

Flowcell with micro-IDEs (250–500 μm) covered with both stable and reverse transfected cells overexpressing membrane receptors to demonstrate impedance responses to serial injections of analyte.

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 Human G protein-coupled receptor 30 is N-glycosylated and N-terminal domain asparagine 44 is required for receptor structure and activity

2019 ◽  
Vol 39 (2) ◽  
Author(s):  
Ernesto Gonzalez de Valdivia ◽  
Caroline Sandén ◽  
Robin Kahn ◽  
Björn Olde ◽  
L.M. Fredrik Leeb-Lundberg
Keyword(s):  
Estrogen Receptor ◽  
G Protein ◽  
Hek293 Cells ◽  
G Protein Coupled Receptor ◽  
Hek 293 ◽  
Receptor Structure ◽  
Terminal Domain ◽  
Endocytic Vesicles ◽  
G Protein Coupled ◽  
Structure And Activity

Abstract G protein-coupled receptor 30 (GPR30), or G protein-coupled estrogen receptor (GPER), is a G protein-coupled receptor (GPCR) that is currently attracting considerable attention in breast cancer and cardiometabolic regulation. The receptor was reported to be a novel membrane estrogen receptor mediating rapid non-genomic responses. However, questions remain about both the cognate ligand and the subcellular localization of receptor activity. Here, we used human embryonic kidney (HEK) 293 (HEK293) cells ectopically expressing N-terminally FLAG-tagged human GPR30 and three unique antibodies (Ab) specifically targetting the receptor N-terminal domain (N-domain) to investigate the role of N-glycosylation in receptor maturation and activity, the latter assayed by constitutive receptor-stimulated extracellular-regulated protein kinase (ERK) 1/2 (ERK1/2) activity. GPR30 expression was complex with receptor species spanning from approximately 40 kDa to higher molecular masses and localized in the endoplasmatic reticulum (ER), the plasma membrane (PM), and endocytic vesicles. The receptor contains three conserved asparagines, Asn25, Asn32, and Asn44, in consensus N-glycosylation motifs, all in the N-domain, and PNGase F treatment showed that at least one of them is N-glycosylated. Mutating Asn44 to isoleucine inactivated the receptor, yielding a unique receptor species at approximately 20 kDa that was recognized by Ab only in a denatured state. On the other hand, mutating Asn25 or Asn32 either individually or in combination, or truncating successively N-domain residues 1–42, had no significant effect either on receptor structure, maturation, or activity. Thus, Asn44 in the GPR30 N-domain is required for receptor structure and activity, whereas N-domain residues 1–42, including specifically Asn25 and Asn32, do not play any major structural or functional role(s).

scholarly journals The G protein coupled receptor CXCR4 designed by the QTY code becomes more hydrophilic and retains cell signaling activity

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Lotta Tegler ◽  
Karolina Corin ◽  
Horst Pick ◽  
Jennifer Brookes ◽  
Michael Skuhersky ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Membrane Protein ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
Hek293 Cells ◽  
Biological Functions ◽  
Wide Range ◽  
Medicinal Drugs ◽  
G Protein Coupled ◽  
Signaling Activity

AbstractG protein-coupled receptors (GPCRs) are vital for diverse biological functions, including vision, smell, and aging. They are involved in a wide range of diseases, and are among the most important targets of medicinal drugs. Tools that facilitate GPCR studies or GPCR-based technologies or therapies are thus critical to develop. Here we report using our QTY (glutamine, threonine, tyrosine) code to systematically replace 29 membrane-facing leucine, isoleucine, valine, and phenylalanine residues in the transmembrane α-helices of the GPCR CXCR4. This variant, CXCR4QTY29, became more hydrophilic, while retaining the ability to bind its ligand CXCL12. When transfected into HEK293 cells, it inserted into the cell membrane, and initiated cellular signaling. This QTY code has the potential to improve GPCR and membrane protein studies by making it possible to design functional hydrophilic receptors. This tool can be applied to diverse α-helical membrane proteins, and may aid in the development of other applications, including clinical therapies.

scholarly journals Smoothened determines β-arrestin–mediated removal of the G protein–coupled receptor Gpr161 from the primary cilium

2016 ◽  
Vol 212 (7) ◽  
pp. 861-875 ◽  
Author(s):  
Kasturi Pal ◽  
Sun-hee Hwang ◽  
Bandarigoda Somatilaka ◽  
Hemant Badgandi ◽  
Peter K. Jackson ◽  
...  
Keyword(s):  
Membrane Protein ◽  
G Protein ◽  
Primary Cilium ◽  
Protein Composition ◽  
Negative Regulator ◽  
G Protein Coupled Receptor ◽  
New Paradigm ◽  
Subcellular Compartment ◽  
Shh Pathway ◽  
G Protein Coupled

Dynamic changes in membrane protein composition of the primary cilium are central to development and homeostasis, but we know little about mechanisms regulating membrane protein flux. Stimulation of the sonic hedgehog (Shh) pathway in vertebrates results in accumulation and activation of the effector Smoothened within cilia and concomitant disappearance of a negative regulator, the orphan G protein–coupled receptor (GPCR), Gpr161. Here, we describe a two-step process determining removal of Gpr161 from cilia. The first step involves β-arrestin recruitment by the signaling competent receptor, which is facilitated by the GPCR kinase Grk2. An essential factor here is the ciliary trafficking and activation of Smoothened, which by increasing Gpr161–β-arrestin binding promotes Gpr161 removal, both during resting conditions and upon Shh pathway activation. The second step involves clathrin-mediated endocytosis, which functions outside of the ciliary compartment in coordinating Gpr161 removal. Mechanisms determining dynamic compartmentalization of Gpr161 in cilia define a new paradigm for down-regulation of GPCRs during developmental signaling from a specialized subcellular compartment.

Internalization of the M2 muscarinic acetylcholine receptor proceeds through an atypical pathway in HEK293 cells that is independent of clathrin and caveolae

2001 ◽  
Vol 114 (4) ◽  
pp. 739-746 ◽  
Author(s):  
A.G. Roseberry ◽  
M.M. Hosey
Keyword(s):  
G Protein ◽  
Acetylcholine Receptor ◽  
Muscarinic Acetylcholine Receptor ◽  
Hek293 Cells ◽  
G Protein Coupled Receptor ◽  
Coated Pits ◽  
Muscarinic Acetylcholine ◽  
G Protein Coupled

The M2 muscarinic acetylcholine receptor is a G-protein coupled receptor that undergoes agonist-induced internalization through an unidentified pathway that exhibits an atypical dependence on dynamin function in HEK293 cells. In this report we utilized several independent approaches to reveal that the internalization of the M2 muscarinic acetylcholine receptor did not utilize clathrin-coated pits or caveolae. However, we did observe that treatment with hypertonic sucrose, which is widely reported to specifically inhibit endocytosis through clathrin-coated pits, completely inhibited internalization of the M2 muscarinic acetylcholine receptor. Thus, the pathway that mediates the internalization of the M2 muscarinic acetylcholine receptor appears to be atypical in that it exhibits an unusual sensitivity to dynamin and is inhibited by hypertonic sucrose but lacks the involvement of clathrin and caveolae.

Contribution of endogenous G-protein-coupled receptor kinases to Ser129 phosphorylation of α-synuclein in HEK293 cells

2009 ◽  
Vol 384 (3) ◽  
pp. 378-382 ◽  
Author(s):  
Masahiro Sakamoto ◽  
Shigeki Arawaka ◽  
Susumu Hara ◽  
Hiroyasu Sato ◽  
Can Cui ◽  
...  
Keyword(s):  
G Protein ◽  
Hek293 Cells ◽  
G Protein Coupled Receptor ◽  
Receptor Kinases ◽  
G Protein Coupled

scholarly journals Regulation of ovarian cancer G protein-coupled receptor-1 expression and signaling

2019 ◽  
Vol 316 (5) ◽  
pp. L894-L902 ◽  
Author(s):  
Ajay P. Nayak ◽  
Tonio Pera ◽  
Deepak A. Deshpande ◽  
James V. Michael ◽  
Jennifer R. Liberato ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
G Protein ◽  
Chronic Treatment ◽  
Cardiac Tissue ◽  
Model Systems ◽  
Hek293 Cells ◽  
Multiple Model ◽  
Heterotrimeric G Proteins ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

Ovarian cancer G protein-coupled receptor 1 (OGR1) is a recently deorphanized G protein-coupled receptor shown to signal in response to low extracellular pH (↓pHo) or certain benzodiazepines. The pleiotropic nature of OGR1 signaling in human airway smooth muscle (HASM) cells suggests that OGR1 is a potential therapeutic target for the management of obstructive lung diseases. However, the basic pharmacological and regulatory features of OGR1 remain poorly understood. We employed model systems of heterologously expressed [human embryonic kidney 293 (HEK293) cells] or endogenous (HASM) OGR1 to assess changes in expression, subcellular localization, and signaling capabilities following acute or chronic treatment with ↓pHo or the benzodiazepines lorazepam and sulazepam. In HEK293 cells expressing OGR1, treatment with ↓pHo and/or lorazepam, but not sulazepam, caused rapid OGR1 internalization. In HASM cells, acute treatment with ↓pHo or benzodiazepines did not alter abundance of OGR1 mRNA; however, significant downregulation was observed following chronic treatment. Acute and chronic pretreatment of HASM cells with sulazepam or lorazepam resulted in receptor desensitization as demonstrated by reduced phosphorylation of vasodilator-stimulated phosphoprotein (VASP) or p42/p44 upon rechallenge. Acid (acute but not chronic) pretreatment of HASM cells induced desensitization of OGR1-mediated VASP (but not p42/p44) phosphorylation. In contrast to a recent study reporting OGR1 upregulation and sensitization in cardiac tissue subject to ischemic/acidic insult, chronic OGR1 activation in multiple model systems did not increase OGR1 expression or signaling capacity. The ability to induce OGR1 internalization and desensitization was activator dependent, reflecting the ability of different activators to induce specific receptor confirmations and engagement of specific heterotrimeric G proteins.

scholarly journals Estrogen Signaling Characteristics of Atlantic Croaker G Protein-Coupled Receptor 30 (GPR30) and Evidence It Is Involved in Maintenance of Oocyte Meiotic Arrest

Endocrinology ◽  
2008 ◽  
Vol 149 (7) ◽  
pp. 3410-3426 ◽  
Author(s):  
Yefei Pang ◽  
Jing Dong ◽  
Peter Thomas
Keyword(s):  
G Protein ◽  
Plasma Membranes ◽  
Critical Role ◽  
Hek293 Cells ◽  
Atlantic Croaker ◽  
Estrogen Signaling ◽  
G Protein Coupled Receptor ◽  
Meiotic Arrest ◽  
Transfected Cells ◽  
G Protein Coupled

Human G protein-coupled receptor 30 (GPR30) mediates estradiol-17β (E2) activation of adenylyl cyclase in breast cancer cells and displays E2 binding typical of membrane estrogen receptors (mERs). We identified a mER in Atlantic croaker ovaries with characteristics similar to those of human GPR30. To confirm the proposed role of GPR30 as a mER in this distantly related vertebrate group, we cloned GPR30 from croaker ovaries and examined its distribution, steroid binding, and signaling characteristics. Western blot analysis showed the GPR30 protein (∼40 kDa) is expressed on the plasma membranes of croaker oocytes and HEK293 cells stably transfected with GPR30 cDNA. Plasma membranes prepared from croaker GPR30-transfected cells displayed high-affinity, limited-capacity, and displaceable binding specific for estrogens, characteristic of mERs. Consistent with previous findings with human GPR30, estrogen treatment of plasma membranes from both croaker ovaries and GPR30-transfected cells caused activation of a stimulatory G protein (Gs) resulting in increased cAMP production. Treatment with E2 as well as G-1, a specific GPR30 ligand, significantly reduced both spontaneous and progestin-induced maturation of both croaker and zebrafish oocytes in vitro, suggesting a possible involvement of GPR30 in maintaining oocyte meiotic arrest in these species. Injection of antisense oligonucleotides to GPR30 into zebrafish oocytes blocked the inhibitory effects of estrogen on oocyte maturation, confirming a role for GPR30 in the control of meiotic arrest. These findings further support our previous suggestion that GPR30 is a vertebrate mER. In addition, the results suggest GRP30 may play a critical role in regulating reentry into the meiotic cell cycle in fish oocytes.

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