Emerging roles of a chemoattractant receptor GPR15 and ligands in pathophysiology.

Chemokine receptors play a central role in the maintenance of immune homeostasis and development of inflammation by directing leukocyte migration to tissues. GPR15 is a G protein-coupled receptor (GPCR) that was initially known as a co-receptor for human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), with structural similarity to other members of the chemoattractant receptor family. Since the discovery of its novel function as a colon-homing receptor of T cells in mice a decade ago, GPR15 has been rapidly gaining attention for its involvement in a variety of inflammatory and immune disorders.

Dual function of Rab1A in secretion and autophagy: hypervariable domain dependence.

We currently understand how the different intracellular pathways, secretion, endocytosis, and autophagy are regulated by small GTPases. In contrast, it is unclear how these pathways are coordinated to ensure efficient cellular response to stress. Rab GTPases localize to specific organelles through their hypervariable domain (HVD) to regulate discrete steps of individual pathways.

Tyrosine sulfation and O-glycosylation of chemoattractant receptor GPR15 differentially regulate interaction with GPR15L.

GPR15 is a G-protein-coupled receptor (GPCR) that directs lymphocyte homing to the colon and skin. Recent studies have identified a chemokine-like protein GPR15L (also known as C10orf99) as a functional ligand of GPR15. In this study, we examined the structural elements that regulate the GPR15-GPR15L interaction with primary focus on post-translational modifications (PTMs) of receptor N-terminus and on the C-terminus of the ligand.

Biased antagonism of CXCR4 avoids antagonist tolerance.

Repeated dosing of drugs targeting G protein-coupled receptors can stimulate antagonist tolerance, which reduces their efficacy; thus, strategies to avoid tolerance are needed. The efficacy of AMD3100, a competitive antagonist of the chemokine receptor CXCR4 that mobilizes leukemic blasts from the bone marrow into the blood to sensitize them to chemotherapy, is reduced after prolonged treatment. Tolerance to AMD3100 increases the abundance of CXCR4 on the surface of leukemic blasts, which promotes their rehoming to the bone marrow.

Positive zip coding in small protein translocation.

Most newly synthesized proteins destined for the secretory pathway contain a signal peptide (SP) that triggers cotranslational translocation into the endoplasmic reticulum (ER). However, how small polypeptides undergo ER translocation is not fully understood. In this issue of , Guo describe a mechanism for posttranslational translocation of small secretory proteins featuring a positive charge within the SP N-terminal region.

Differential phosphorylation signals control endocytosis of GPR15.

GPR15 is an orphan G protein-coupled receptor (GPCR) that serves for an HIV coreceptor and was also recently found as a novel homing receptor for T-cells implicated in colitis. We show that GPR15 undergoes a constitutive endocytosis in the absence of ligand. The endocytosis was clathrin dependent and partially dependent on β-arrestin in HEK293 cells, and nearly half of the internalized GPR15 receptors were recycled to the plasma membrane.

Potential use of potassium efflux-deficient yeast for studying trafficking signals and potassium channel functions.

The activity of potassium (K(+)) channels critically depends on their density on the cell surface membrane, which is regulated by dynamic protein-protein interactions that often involve distinct trafficking signals on the cargo proteins. In this paper we explored the possibility of utilizing the Saccharomyces cerevisiae strain B31 for identification of the signal motifs that regulate surface expression of membrane proteins and for studying structure-function relationships of K(+) channels. B31 cells lack the K(+) efflux system and were reported to show overloaded K(+)-mediated growth inhibition in high K(+) media upon heterologous expression of a mammalian inwardly rectifying K(+) channel (Kir2.

Role of C-terminal membrane-proximal basic residues in cell surface trafficking of HIV coreceptor GPR15 protein.

Cell surface density of G protein-coupled receptors (GPCRs) is controlled by dynamic molecular interactions that often involve recognition of the distinct sequence signals on the cargo receptors. We reported previously that the RXR-type dibasic motif in the distal C-terminal tail of an HIV coreceptor GPR15 negatively regulates the cell surface expression by mediating the coatomer protein I complex-dependent retrograde transport to the endoplasmic reticulum (ER). Here we demonstrate that another pair of basic residues (Arg(310)-Arg(311)) in the membrane-proximal region of the C-terminal tail plays a pivotal role in mediating the anterograde trafficking of GPR15.

Up-regulation of A-type potassium currents protects neurons against cerebral ischemia.

Excitotoxicity is the major cause of many neurologic disorders including stroke. Potassium currents modulate neuronal excitability and therefore influence the pathological process. A-type potassium current (I(A)) is one of the major voltage-dependent potassium currents, yet its roles in excitotoxic cell death are not well understood.

Phosphorylation-dependent C-terminal binding of 14-3-3 proteins promotes cell surface expression of HIV co-receptor GPR15.

Membrane trafficking is dictated by dynamic molecular interactions involving discrete determinants in the cargo proteins and the intracellular transport machineries. We have previously reported that cell surface expression of GPR15, a G protein-coupled receptor (GPCR) that serves as a co-receptor for HIV, is correlated with the mode III binding of 14-3-3 proteins to the receptor C terminus. Here we provide a mechanistic basis for the role of 14-3-3 in promoting the cell surface expression of GPR15.

PI3K/Akt signalling-mediated protein surface expression sensed by 14-3-3 interacting motif.

The regulation of protein expression on the cell surface membrane is an important component of the cellular response to extracellular signalling. The translation of extracellular signalling into specific protein localization often involves the post-translational modification of cargo proteins. Using a genetic screen of random peptides, we have previously identified a group of C-terminal sequences, represented by RGRSWTY-COOH (termed'SWTY'), which are capable of overriding an endoplasmic reticulum localization signal and directing membrane proteins to the cell surface via specific binding to 14-3-3 proteins.

Differential trafficking of carboxyl isoforms of Ca2+-gated (Slo1) potassium channels.

The pore-forming subunit of the large-conductance Ca(2+)-dependent K(+) (Slo1) channel is encoded by one gene. However, the functional properties of Slo1 channels are diverse in part because of their numerous regulatory mechanisms including posttranslational modification and alternative splicing. In particular, multiple splice variants of the pore-forming subunit have been reported but their significance is only beginning to be elucidated.

Phospho-specific recognition by 14-3-3 proteins and antibodies monitored by a high throughput label-free optical biosensor.

Label-free detection of molecular interactions has considerable potential in facilitating assay development. When combined with high throughput capability, it may be applied to small molecule screens for drug candidates. Phosphorylation is a key posttranslational process that confers diverse regulation in biological systems involving specific protein-protein interactions recognizing the phosphorylated motifs.

14-3-3 proteins: regulation of endoplasmic reticulum localization and surface expression of membrane proteins.

The density and composition of cell surface proteins are major determinants for cellular functions. Regulation of cell surface molecules occurs at several levels, including the efficiency of surface transport, and is therefore of great interest. As the major phosphoprotein-binding modules, 14-3-3 proteins are known for their crucial roles in a wide range of cellular activities, including the subcellular localization of target proteins.

C-terminal binding: an expanded repertoire and function of 14-3-3 proteins.

Amino and carboxyl termini are unique positions in a polypeptide. They tend to be exposed in folded three dimensional structures. Diversity and functional significance of C-terminal sequences have been appreciated from studies of PDZ and PEX domains.

Chronic inhibition of cardiac Kir2.1 and HERG potassium channels by celastrol with dual effects on both ion conductivity and protein trafficking.

A high percentage of drugs and drug candidates has been found to cause cardiotoxicity by reducing potassium conductance, more commonly known as QT prolongation. However, some compounds do not show direct block of ionic flow, suggesting that other mechanisms may also lead to reduction of potassium currents. Using the functional recovery after chemobleaching (FRAC) assay, we have examined a collection of drugs and drug-like compounds for potential perturbation of cardiac potassium channel trafficking.

SWTY--a general peptide probe for homogeneous solution binding assay of 14-3-3 proteins.

Dimeric 14-3-3 proteins exert diverse functions in eukaryotes by binding to specific phosphorylated sites on diverse target proteins. Critical to the physiological function of 14-3-3 proteins is the wide range of binding affinity to different ligands. The existing information of binding affinity is mainly derived from nonhomogeneous-based methods such as surface plasmon resonance and quantitative affinity precipitation.

Genetic isolation of transport signals directing cell surface expression.

Membrane proteins represent approximately 30% of the proteome in both prokaryotes and eukaryotes. The spatial localization of membrane-bound proteins is often determined by specific sequence motifs that may be regulated in response to physiological changes, such as protein interactions and receptor signalling. Identification of signalling motifs is therefore important for understanding membrane protein expression, function and transport mechanisms.

Selective expression of vacuolar H+-ATPase subunit d2 by particular subsets of dendritic cells among leukocytes.

Dendritic cells (DC) are far more potent to activate T cells than other antigen presenting cells (e.g., macrophages) and distributed to many organs where DC develop to functionally and phenotypically distinctive subsets.

C-terminal recognition by 14-3-3 proteins for surface expression of membrane receptors.

Diverse functions of 14-3-3 proteins are directly coupled to their ability to interact with targeted peptide substrates. RSX(pS/pT)XP and RXPhiX(pS/pT)XP are two canonical consensus binding motifs for 14-3-3 proteins representing the two common binding modes, modes I and II, between 14-3-3 and internal peptides. Using a genetic selection, we have screened a random peptide library and identified a group of C-terminal motifs, termed SWTY, capable of overriding an endoplasmic reticulum localization signal and redirecting membrane proteins to cell surface.

Function recovery after chemobleaching (FRAC): evidence for activity silent membrane receptors on cell surface.

Membrane proteins represent approximately 30% of the proteome of both prokaryotes and eukaryotes. Unique to cell surface receptors is their biogenesis pathway, which involves vesicular trafficking from the endoplasmic reticulum through the Golgi apparatus and to the cell surface. Increasing evidence suggests specific regulation of biogenesis for different membrane receptors, hence affecting their surface expression.

Membrane receptor trafficking: evidence of proximal and distal zones conferred by two independent endoplasmic reticulum localization signals.

The generic membrane trafficking signals of internal RXR and carboxyl-terminal KKXX motifs direct intracellular endoplasmic reticulum (ER) localization of the signal-bearing proteins. These signaling motifs play a critical role in partitioning proteins into designated subcellular compartments by functioning as an intracellular "zip code." In the process of determining the potential distinctions between these two otherwise functionally identical motifs, two functional zones of these signals were revealed.

Functional diversity of protein C-termini: more than zipcoding?

The carboxylated (C)-terminus of proteins, which includes the single terminal alpha-carboxyl group and preceding residues, is uniquely positioned to serve as a recognition signature for a variety of cell-biological processes, including protein targeting, subcellular anchoring and the static and dynamic formation of macromolecular complexes. The terminal sequence motifs can be processed by posttranslational modifications, thereby providing a means to increase sequence diversity and to regulate interactions. Several classes of protein domains have been identified that are either designed for or are capable of interacting with protein C-termini - these include PDZ and TPR domains.