scholarly journals Functional Characterization of the obesity-linked variant of the β3-adrenergic receptor

2021 ◽  
Author(s):  
Esraa Haji ◽  
Saeed Al Mahri ◽  
Yumna Aloraij ◽  
Shuja Shafi Malik ◽  
Sameer Mohammad
Keyword(s):  
Cell Surface ◽  
Adrenergic Receptor ◽  
Functional Characterization ◽  
Biochemical Properties ◽  
Cell Surface Expression ◽  
Cellular Distribution ◽  
Hek 293 ◽  
Regulatory Pathways ◽  
Beta 2 ◽  
Activation Behavior

Adrenergic receptor β3 (ADRβ3) is a member of the rhodopsin-like G protein-coupled receptor family. The binding of the ligand to ADRβ3 activates adenylate cyclase and increases cAMP in the cells. ADRβ3 is highly expressed in white and brown adipocytes and controls key regulatory pathways of lipid metabolism. Trp64Arg (W64R) polymorphism in the ADRβ3 has been associated with the early development of type 2 diabetes mellitus, lower resting metabolic rate, abdominal obesity, and insulin resistance. It is unclear how the substitution of W64R affects the functioning of ADRβ3. This study was initiated to functionally characterize this obesity-linked variant of ADRβ3. We evaluated in detail the expression, subcellular distribution, and post-activation behavior of the WT and W64R ADRβ3 using a single cell quantitative fluorescence microscopy. When expressed in HEK 293 cells, ADRβ3 shows a typical distribution displayed by other GPCRs with a predominant localization at the cell surface. Unlike Adrenergic receptor β2 (ADRβ2), agonist induced desensitization of ADRβ3 does not involve loss of cell surface expression. WT and W64R variant of ADRβ3 displayed comparable biochemical properties and there was no significant impact of the substitution of Tryptophan with Arginine on the expression, cellular distribution, signaling, and post-activation behavior of ADRβ3. The obesity-linked W64R variant of ADRβ3 is indistinguishable from the WT ADRβ3 in terms of expression, cellular distribution, signaling, and post-activation behavior.

scholarly journals Functional Characterization of the Obesity-Linked Variant of the β3-Adrenergic Receptor

2021 ◽  
Vol 22 (11) ◽  
pp. 5721
Author(s):  
Esraa Haji ◽  
Saeed Al Mahri ◽  
Yumna Aloraij ◽  
Shuja Shafi Malik ◽  
Sameer Mohammad
Keyword(s):  
Cell Surface ◽  
Adrenergic Receptor ◽  
Resting Metabolic Rate ◽  
Functional Characterization ◽  
Biochemical Properties ◽  
Cell Surface Expression ◽  
Cellular Distribution ◽  
Hek 293 ◽  
Regulatory Pathways ◽  
Activation Behavior

Adrenergic receptor β3 (ADRβ3) is a member of the rhodopsin-like G protein-coupled receptor family. The binding of the ligand to ADRβ3 activates adenylate cyclase and increases cAMP in the cells. ADRβ3 is highly expressed in white and brown adipocytes and controls key regulatory pathways of lipid metabolism. Trp64Arg (W64R) polymorphism in the ADRβ3 is associated with the early development of type 2 diabetes mellitus, lower resting metabolic rate, abdominal obesity, and insulin resistance. It is unclear how the substitution of W64R affects the functioning of ADRβ3. This study was initiated to functionally characterize this obesity-linked variant of ADRβ3. We evaluated in detail the expression, subcellular distribution, and post-activation behavior of the WT and W64R ADRβ3 using single cell quantitative fluorescence microscopy. When expressed in HEK 293 cells, ADRβ3 shows a typical distribution displayed by other GPCRs with a predominant localization at the cell surface. Unlike adrenergic receptor β2 (ADRβ2), agonist-induced desensitization of ADRβ3 does not involve loss of cell surface expression. WT and W64R variant of ADRβ3 displayed comparable biochemical properties, and there was no significant impact of the substitution of tryptophan with arginine on the expression, cellular distribution, signaling, and post-activation behavior of ADRβ3. The obesity-linked W64R variant of ADRβ3 is indistinguishable from the WT ADRβ3 in terms of expression, cellular distribution, signaling, and post-activation behavior.

scholarly journals Palmitoylation is not required for trafficking of human anion exchanger 1 to the cell surface

2004 ◽  
Vol 378 (3) ◽  
pp. 1015-1021 ◽  
Author(s):  
Joanne C. CHEUNG ◽  
Reinhart A. F. REITHMEIER
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Palmitic Acid ◽  
Anion Exchanger ◽  
Cell Surface Expression ◽  
Co2 Removal ◽  
Anion Exchanger 1 ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

AE1 (anion exchanger 1) is a glycoprotein found in the plasma membrane of erythrocytes, where it mediates the electroneutral exchange of chloride and bicarbonate, a process important in CO2 removal from tissues. It had been previously shown that human AE1 purified from erythrocytes is covalently modified at Cys-843 in the membrane domain with palmitic acid. In this study, the role of Cys-843 in human AE1 trafficking was investigated by expressing various AE1 and Cys-843Ala (C843A) mutant constructs in transiently transfected HEK-293 cells. The AE1 C843A mutant was expressed to a similar level to AE1. The rate of N-glycan conversion from high-mannose into complex form in a glycosylation mutant (N555) of AE1 C843A, and thus the rate of trafficking from the endoplasmic reticulum to the Golgi, were comparable with that of AE1 (N555). Like AE1, AE1 C843A could be biotinylated at the cell surface, indicating that a cysteine residue at position 843 is not required for cell-surface expression of the protein. The turnover rate of AE1 C843A was not significantly different from AE1. While other proteins could be palmitoylated, labelling of transiently transfected HEK-293 cells or COS7 cells with [3H]palmitic acid failed to produce any detectable AE1 palmitoylation. These results suggest that AE1 is not palmitoylated in HEK-293 or COS7 cells and can traffic to the plasma membrane.

Abstract 049: Functional Characterization of KCNQ1 Channel Subunit With an A590T Mutation

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Koshi Kinoshita ◽  
Katsuya Kimoto ◽  
Takuto Komatsu ◽  
Kohki Nishide ◽  
Toshihide Tabata ◽  
...  
Keyword(s):  
Cell Surface ◽  
Functional Characterization ◽  
Coiled Coil ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Delayed Rectifier ◽  
Wild Type ◽  
Cardiac Events ◽  
Channel Function ◽  
Similar Density

Background: KCNQ1 encodes the alpha subunit of the voltage-gated K + channel that mediates the cardiac slow delayed rectifier K + current (I Ks ). A mutation, A590T, in KCNQ1 was incidentally identified in a 40 years old female. She had a mild QTc prolongation in electrocardiogram but has never experienced any cardiac events. A590 is located in the C-terminal domain forming a coiled-coil structure, which has been suggested as a pivotal component for subunit tetramerization and channel trafficking to the cell surface. The previously reported mutations around A590 result in markedly reduced cell surface expression and loss of functional channel. We, for the first time, examined whether and how the A590T mutation affects the I Ks channel function. Methods: To assess the trafficking and channel function of KCNQ1(A590T) mutant subunit, we performed immunostaining, immunoblotting, and voltage-clamp measurements in HEK-293T cells transfected with wild-type or the A590T mutant KCNQ1 or their mixture (WT, A590T, and A590T/WT cells, respectively). Results: The density of a depolarization-activated current in the A590T cells was smaller than that in the WT cells. The threshold, half-maximal activation, and saturating voltages of the depolarization-activated current in the A590T cells were more positive than those in the WT cells. The immunoreactivity against KCNQ1 subunit on the cell surface in the A590T cells is lower than in WT cells. The A590T/WT cells had a similar density of the depolarization-activated current and a similar level of immunoreactivity against the channel subunit to the WT cells. Furthermore, the immunoblotting detected subunit oligomers in the membrane fraction of the A590T cells while their densities were lower than those of the WT cells. Conclusion: Although the A590T mutant subunit can form oligomers for itself, this subunit is not efficiently trafficked to the cell surface without the aid of the WT subunit. Thus, homozygous inheritance of the mutant KCNQ1 might be pathogenic. By contrast, the cells expressing both the mutant and wild-type KCNQ1 subunit had normal I Ks and cell surface expression, indicating that the heterozygous inheritance of the mutant KCNQ1 might not cause severe cardiac diseases.

Vasopressin-inducible ubiquitin-specific protease 10 increases ENaC cell surface expression by deubiquitylating and stabilizing sorting nexin 3

2008 ◽  
Vol 295 (4) ◽  
pp. F889-F900 ◽  
Author(s):  
Sheerazed Boulkroun ◽  
Dorothée Ruffieux-Daidié ◽  
Jean-Jacques Vitagliano ◽  
Olivier Poirot ◽  
Roch-Philippe Charles ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Long Term Effects ◽  
Hek 293 ◽  
Fourfold Increase ◽  
Sorting Nexin ◽  
Cell Surface Biotinylation ◽  
Specific Protease ◽  
Ubiquitin Specific Protease

Adjustment of Na+ balance in extracellular fluids is achieved by regulated Na+ transport involving the amiloride-sensitive epithelial Na+ channel (ENaC) in the distal nephron. In this context, ENaC is controlled by a number of hormones, including vasopressin, which promotes rapid translocation of water and Na+ channels to the plasma membrane and long-term effects on transcription of vasopressin-induced and -reduced transcripts. We have identified a mRNA encoding the deubiquitylating enzyme ubiquitin-specific protease 10 (Usp10), whose expression is increased by vasopressin at both the mRNA and the protein level. Coexpression of Usp10 in ENaC-transfected HEK-293 cells causes a more than fivefold increase in amiloride-sensitive Na+ currents, as measured by whole cell patch clamping. This is accompanied by a three- to fourfold increase in surface expression of α- and γ-ENaC, as shown by cell surface biotinylation experiments. Although ENaC is well known to be regulated by its direct ubiquitylation, Usp10 does not affect the ubiquitylation level of ENaC, suggesting an indirect effect. A two-hybrid screen identified sorting nexin 3 (SNX3) as a novel substrate of Usp10. We show that it is a ubiquitylated protein that is degraded by the proteasome; interaction with Usp10 leads to its deubiquitylation and stabilization. When coexpressed with ENaC, SNX3 increases the channel's cell surface expression, similarly to Usp10. In mCCDcl1 cells, vasopressin increases SNX3 protein but not mRNA, supporting the idea that the vasopressin-induced Usp10 deubiquitylates and stabilizes endogenous SNX3 and consequently promotes cell surface expression of ENaC.

scholarly journals Mutants of human ACE2 differentially promote SARS-CoV and SARS-CoV-2 spike mediated infection

2021 ◽  
Vol 17 (7) ◽  
pp. e1009715
Author(s):  
Nidhi Shukla ◽  
Sarah M. Roelle ◽  
Vinicius G. Suzart ◽  
Anna M. Bruchez ◽  
Kenneth A. Matreyek
Keyword(s):  
Cell Surface ◽  
293T Cell ◽  
Surface Expression ◽  
Biochemical Properties ◽  
Reference Sequence ◽  
Cell Surface Expression ◽  
Target Cells ◽  
Interaction Interface ◽  
Binding Avidity ◽  
Spike Proteins

SARS-CoV and SARS-CoV-2 encode spike proteins that bind human ACE2 on the cell surface to enter target cells during infection. A small fraction of humans encode variants of ACE2, thus altering the biochemical properties at the protein interaction interface. These and other ACE2 coding mutants can reveal how the spike proteins of each virus may differentially engage the ACE2 protein surface during infection. We created an engineered HEK 293T cell line for facile stable transgenic modification, and expressed the major human ACE2 allele or 28 of its missense mutants, 24 of which are possible through single nucleotide changes from the human reference sequence. Infection with SARS-CoV or SARS-CoV-2 spike pseudotyped lentiviruses revealed that high ACE2 cell-surface expression could mask the effects of impaired binding during infection. Drastically reducing ACE2 cell surface expression revealed a range of infection efficiencies across the panel of mutants. Our infection results revealed a non-linear relationship between soluble SARS-CoV-2 RBD binding to ACE2 and pseudovirus infection, supporting a major role for binding avidity during entry. While ACE2 mutants D355N, R357A, and R357T abrogated entry by both SARS-CoV and SARS-CoV-2 spike proteins, the Y41A mutant inhibited SARS-CoV entry much more than SARS-CoV-2, suggesting differential utilization of the ACE2 side-chains within the largely overlapping interaction surfaces utilized by the two CoV spike proteins. These effects correlated well with cytopathic effects observed during SARS-CoV-2 replication in ACE2-mutant cells. The panel of ACE2 mutants also revealed altered ACE2 surface dependencies by the N501Y spike variant, including a near-complete utilization of the K353D ACE2 variant, despite decreased infection mediated by the parental SARS-CoV-2 spike. Our results clarify the relationship between ACE2 abundance, binding, and infection, for various SARS-like coronavirus spike proteins and their mutants, and inform our understanding for how changes to ACE2 sequence may correspond with different susceptibilities to infection.

scholarly journals Increase in positive selection of CD8+ T cells in TAP1-mutant mice by human beta 2-microglobulin transgene.

1995 ◽  
Vol 181 (2) ◽  
pp. 787-792 ◽  
Author(s):  
H Martien van Santen ◽  
A Woolsey ◽  
P G Rickardt ◽  
L Van Kaer ◽  
E J Baas ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
Mhc Class I ◽  
Cd8 T Cells ◽  
Surface Expression ◽  
Class I ◽  
Cell Surface Expression ◽  
Beta 2 ◽  
Beta 2 Microglobulin ◽  
Selection Of

Mice harboring a deletion of the gene encoding the transporter associated with antigen presentation-1 (TAP1) are impaired in providing major histocompatibility complex (MHC) class I molecules with peptides of cytosolic origin and lack stable MHC class I cell surface expression. They consequently have a strongly reduced number of CD8+ T cells. To examine whether selection of CD8+ T cells is dependent on TAP-dependent peptides, we partially restored MHC class I cell surface expression in TAP1-deficient mice by introduction of human beta 2-microglobulin. We show that selection of functional CD8+ T cells can be augmented in vivo in the absence of TAP1-dependent peptides.

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