scholarly journals Two Domains of the Erythropoietin Receptor Are Sufficient for Jak2 Binding/Activation and Function

2006 ◽  
Vol 26 (22) ◽  
pp. 8527-8538 ◽  
Author(s):  
Stéphane Pelletier ◽  
Sébastien Gingras ◽  
Megumi Funakoshi-Tago ◽  
Sherié Howell ◽  
James N. Ihle
Keyword(s):  
Signal Transduction ◽  
Biological Activity ◽  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Surface Expression ◽  
Proximal Region ◽  
Erythropoietin Receptor ◽  
Cell Surface Expression ◽  
And Function ◽  
Mutant Receptors

ABSTRACT Biochemical and genetic studies have shown that Jak2 is an essential component of EpoR signal transduction which is required for normal erythropoiesis. However, whether Jak2 is the sole direct mediator of EpoR signal transduction remains controversial. To address this issue, we have used an extensive and systematic mutational analysis across the EpoR cytoplasmic tail and transmembrane domain with the goal of determining whether mutants that negatively affected EpoR biological activity but retained Jak2 activation could be identified. Analysis of over 40 mutant receptors established that two large domains in the membrane-proximal region, which include the previously defined Box1 and Box2 domains as well as a highly conserved glycine among cytokine receptors, are required for Jak2 binding and activation and to sustain biological activity of the receptor. Importantly, none of the mutants that lost the ability to activate Jak2 retained the ability to bind Jak2, thus questioning the validity of models of receptor reorientation for Jak2 activation. Also, no correlation was made between cell surface expression of the receptor and its ability to bind Jak2, thus questioning the role of Jak2 in trafficking the receptor to the plasma membrane. Collectively, the results suggest that Jak2 is the sole direct signaling molecule downstream of EpoR required for biological activity.

scholarly journals Atypical Fusion Peptide of Nelson Bay Virus Fusion-Associated Small Transmembrane Protein

2005 ◽  
Vol 79 (3) ◽  
pp. 1853-1860 ◽  
Author(s):  
LiTing T. Cheng ◽  
Richard K. Plemper ◽  
Richard W. Compans
Keyword(s):  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Transmembrane Protein ◽  
Syncytium Formation ◽  
Surface Expression ◽  
Fusion Peptide ◽  
Cell Surface Expression ◽  
Type I ◽  
Fusion Activity ◽  
Hydrophobic Domain

ABSTRACT A 10-kDa nonstructural transmembrane protein (p10) encoded by a reovirus, Nelson Bay virus, has been shown to induce syncytium formation (34). Sequence analysis and structural studies identified p10 as a type I membrane protein with a central transmembrane domain, a cytoplasmic basic region, and an N-terminal hydrophobic domain (HD) that was hypothesized to function as a fusion peptide. We performed mutational analysis on this slightly hydrophobic motif to identify possible structural requirements for fusion activity. Bulky aliphatic residues were found to be essential for optimal fusion, and an aromatic or highly hydrophobic side chain was found to be required at position 12. The requirement for hydrophilic residues within the HD was also examined: substitution of 10-Ser or 14-Ser with hydrophobic residues was found to reduce cell surface expression of p10 and delayed the onset of syncytium formation. Nonconservative substitutions of charged residues in the HD did not have an effect on fusion activity. Taken together, our results suggest that the HD is involved in both syncytium formation and in determining p10 transport and surface expression.

scholarly journals Co-translational folding of the first transmembrane domain of ABC-transporter CFTR is supported by assembly with the first cytosolic domain

2020 ◽  
Author(s):  
Bertrand Kleizen ◽  
Marcel van Willigen ◽  
Marjolein Mijnders ◽  
Florence Peters ◽  
Magda Grudniewska ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Activity Cycle ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Type I ◽  
Inherited Disease ◽  
Domain Specific ◽  
Independent Events ◽  
Cytosolic Domains ◽  
And Function

ABSTRACTABC-transporters transport a wealth of molecules across membranes and consist of transmembrane and cytosolic domains. Their activity cycle involves a tightly regulated and concerted domain choreography, with regulation driven by the cytosolic domains, and function by the transmembrane domains. Folding of these polytopic multidomain proteins to their functional state is a challenge for cells, which is mitigated by co-translational and sequential events. We here reveal the first stages of co-translational domain folding and assembly of CFTR, the protein defective in the most abundant rare inherited disease cystic fibrosis, by combining biosynthetic radiolabeling with protease-susceptibility assays and domain-specific antibodies. The most N-terminal domain, TMD1 (transmembrane domain 1), folds both its hydrophobic and soluble helices during translation: the transmembrane helices pack tightly and the cytosolic N- and C-termini assemble with the first cytosolic helical loop ICL1, leaving only ICL2 exposed. This N-C-ICL1 assembly is strengthened by two independent events: i) assembly of ICL1 with the N-terminal subdomain of the next domain, cytosolic NBD1 (nucleotide-binding domain 1); and ii) in the presence of corrector drug VX-809, which rescues cell-surface expression of a range of disease-causing CFTR mutants. Both lead to increased shielding of the CFTR N-terminus, and their additivity implies a different mode of action. Early assembly of NBD1 and TMD1 is essential for CFTR folding and positions both domains for the required assembly with TMD2. Altogether, we have gained insights into this first, nucleating, VX-809-enhanced domain-assembly event during and immediately after CFTR translation, involving structures conserved in type-I ABC exporters.

scholarly journals Signal transduction through Cys-loop receptors is mediated by the nonspecific bumping of closely apposed domains

2021 ◽  
Vol 118 (14) ◽  
pp. e2021016118
Author(s):  
Gisela D. Cymes ◽  
Claudio Grosman
Keyword(s):  
Signal Transduction ◽  
Transmembrane Domain ◽  
Surface Expression ◽  
Relative Orientation ◽  
The Other ◽  
Cell Surface Expression ◽  
Side Chains ◽  
Wild Type ◽  
Successful Design ◽  
Patch Clamp Electrophysiology

One of the most fundamental questions in the field of Cys-loop receptors (pentameric ligand-gated ion channels, pLGICs) is how the affinity for neurotransmitters and the conductive/nonconductive state of the transmembrane pore are correlated despite the ∼60-Å distance between the corresponding domains. Proposed mechanisms differ, but they all converge into the idea that interactions between wild-type side chains across the extracellular–transmembrane-domain (ECD–TMD) interface are crucial for this phenomenon. Indeed, the successful design of fully functional chimeras that combine intact ECD and TMD modules from different wild-type pLGICs has commonly been ascribed to the residual conservation of sequence that exists at the level of the interfacial loops even between evolutionarily distant parent channels. Here, using mutagenesis, patch-clamp electrophysiology, and radiolabeled-ligand binding experiments, we studied the effect of eliminating this residual conservation of sequence on ion-channel function and cell-surface expression. From our results, we conclude that proper state interconversion (“gating”) does not require conservation of sequence—or even physicochemical properties—across the ECD–TMD interface. Wild-type ECD and TMD side chains undoubtedly interact with their surroundings, but the interactions between them—straddling the interface—do not seem to be more important for gating than those occurring elsewhere in the protein. We propose that gating of pLGICs requires, instead, that the overall structure of the interfacial loops be conserved, and that their relative orientation and distance be the appropriate ones for changes in one side to result in changes in the other, in a phenomenon akin to the nonspecific “bumping” of closely apposed domains.

scholarly journals Pleiotropic functions of the transmembrane domain 6 of human melanocortin-4 receptor

2012 ◽  
Vol 49 (3) ◽  
pp. 237-248 ◽  
Author(s):  
Hui Huang ◽  
Ya-Xiong Tao
Keyword(s):  
Cell Surface ◽  
Structure Function ◽  
Transmembrane Domain ◽  
Mapk Pathway ◽  
Surface Expression ◽  
Camp Signaling ◽  
Cell Surface Expression ◽  
Melanocortin 4 Receptor ◽  
Function Relationship ◽  
Relationship Of

The melanocortin-4 receptor (MC4R) is a critical regulator of energy homeostasis and has emerged as a premier target for obesity treatment. Numerous mutations in transmembrane domain 6 (TM6) of MC4R resulting in functional alterations have been identified in obese patients. Several mutagenesis studies also provided some data suggesting the importance of this domain in receptor function. To gain a better understanding of the structure–function relationship of the receptor, we performed alanine-scanning mutagenesis in TM6 to determine the functions of side chains. Of the 31 residues, two were important for cell surface expression, five were indispensable for α-melanocyte-stimulating hormone (α-MSH) and β-MSH binding, and six were important for signaling in the Gs–cAMP–PKA pathway. H264A, targeted normally to the plasma membrane, was undetectable by competitive binding assay and severely defective in basal and stimulated cAMP production and ERK1/2 phosphorylation. Nine mutants had decreased basal cAMP signaling. Seven mutants were constitutively active in cAMP signaling and their basal activities could be inhibited by two MC4R inverse agonists, Ipsen 5i and ML00253764. Five mutants were also constitutively active in the MAPK pathway with enhanced basal ERK1/2 phosphorylation. In summary, our study provided comprehensive data on the structure–function relationship of the TM6 of MC4R. We identified residues that are important for cell surface expression, ligand binding, cAMP generation, and residues for maintaining the WT receptor in active conformation. We also reported constitutive activation of the MAPK pathway and biased signaling. These data will be useful for rationally designing MC4R agonists and antagonists for treatment of eating disorders.

scholarly journals The role of prolines and glycine in the transmembrane domain of LAT

2020 ◽  
Author(s):  
Daniela Glatzová ◽  
Harsha Mavila ◽  
Maria Chiara Saija ◽  
Tomáš Chum ◽  
Lukasz Cwiklik ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Transmembrane Helix ◽  
Helical Structure ◽  
Surface Expression ◽  
Transmembrane Segment ◽  
Proline Residue ◽  
And Function ◽  
The Impact

ABSTRACTLAT is a critical regulator of T cell development and function. It organises signalling events at the plasma membrane. However, the mechanism, which controls LAT localisation at the plasma membrane is not fully understood. Here, we studied the impact of helix-breaking amino acids, two prolines and one glycine, in the transmembrane segment on localisation and function of LAT. Using in silico analysis, confocal and superresolution imaging and flow cytometry we demonstrate that central proline residue destabilises transmembrane helix by inducing a kink. The helical structure and dynamics is further regulated by glycine and another proline residue in the luminal part of LAT transmembrane domain. Replacement of these residues with aliphatic amino acids reduces LAT dependence on palmitoylation for sorting to the plasma membrane. However, surface expression of these mutants is not sufficient to recover function of non-palmitoylated LAT in stimulated T cells. These data indicate that geometry and dynamics of LAT transmembrane segment regulate its localisation and function in immune cells.

scholarly journals Cell Surface Expression and Function of the Macromolecular C1 Complex on the Surface of Human Monocytes

2012 ◽  
Vol 3 ◽  
Author(s):  
Kinga K. Hosszu ◽  
Alisa Valentino ◽  
Yan Ji ◽  
Mara Matkovic ◽  
Lina Pednekar ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Human Monocytes ◽  
And Function
Sign in / Sign up

Export Citation Format

Share Document