GPR37, also known as the parkin-associated endothelin-like receptor (Pael-R), is an orphan G protein-coupled receptor (GPCR) that exhibits poor plasma membrane expression when expressed in most cell types. We sought to find ways to enhance GPR37 trafficking to the cell surface in order to facilitate studies of GPR37 functional activity in heterologous cells. In truncation studies, we found that removing the GPR37 N-terminus (NT) dramatically enhanced the receptor's plasma membrane insertion. Further studies on sequential NT truncations revealed that removal of the first 210 amino acids increased surface expression nearly as much as removal of the entire NT. In studies examining the effects of co-expression of GPR37 with a variety of other GPCRs, we observed significant increases in GPR37 surface expression when the receptor was co-expressed with the adenosine receptor A 2A R or the dopamine receptor D 2 R. Co-immunoprecipitation experiments revealed that full-length GPR37 and, to a greater extent, the truncated GPR37 were capable of robustly associating with D 2 R, resulting in modestly-altered D 2 R affinity for both agonists and antagonists. In studies examining potential interactions of GPR37 with PDZ scaffolds, we observed a specific interaction between GPR37 and syntenin-1, which resulted in a dramatic increase in GPR37 surface expression in HEK-293 cells. These findings reveal three independent approaches -N-terminal truncation, coexpression with other receptors and co-expression with syntenin-1 -by which GPR37 surface trafficking in heterologous cells can be greatly enhanced to facilitate functional studies on this orphan receptor.The orphan G-protein coupled receptor (GPCR) GPR37, also known as the parkin-associated endothelin-like receptor (Pael-R), is highly expressed in the mammalian central nervous system, but its function still remains largely unknown. GPR37 is most closely related to another CNS-enriched orphan receptor known as GPR37-like 1 (GPR37L1), and both orphans share significant sequence homology with the endothelin B receptor and other related peptideactivated GPCRs (1-5). However, none of the mammalian peptides tested so far, including endothelins, bombesin and others, have produced activation of any signaling pathways in heterologous cells or Xenopus oocytes expressing either GPR37 or GPR37L1 (2-5). A peptide called "head activator" (HA), which is derived from the freshwater coelenterate Hydra, has been reported to be capable of activating GPR37 (6), but no peptide equivalent to HA has been definitively identified in vertebrates. Thus, GPR37 and GPR37L1 must still be considered orphan GPCRs at the present time.A major stumbling block impeding progress in understanding the ligand binding and signaling of GPR37 is the receptor's poor trafficking to the plasma membrane in most heterologous cell lines. GPR37 is commonly misfolded and therefore aggregated in the endoplasmic reticulum † This work was supported by the Pharmacological Sciences training grant T32 GM008602, the NIH and the...

Background: PKC␣ contains a unique PDZ ligand motif and is known to promote cellular migration. Results: PKC␣ binds and phosphorylates the scaffold DLG1; both proteins are necessary for cellular migration in non-small cell lung cancer cells. Conclusion: DLG1 coordinates PKC␣ signaling to promote cellular migration. Significance: Control of PKC␣ signaling mediated by scaffolds is crucial to promoting its downstream functions.

Protein kinase D (PKD) transduces an abundance of signals downstream of diacylglycerol production. The mammalian PKD family consists of three isoforms, PKD1, PKD2, and PKD3; of these PKD1 and PKD2 contain PDZ-binding motifs at their carboxyl termini. Here we show that membrane-localized NHERF scaffold proteins provide a nexus for tightly controlled PKD signaling via a PDZ domain interaction. Using a proteomic array containing 96 purified PDZ domains, we have identified the first PDZ domain of NHERF-1 as an interaction partner for the PDZ-binding motifs of both PKD1 and PKD2. A fluorescence resonance energy transfer-based translocation assay reveals a transient association of PKD1 and PKD2 with NHERF-1 in live cells that is triggered by phorbol ester stimulation and, importantly, differs strikingly from the sustained translocation to plasma membrane. Targeting a fluorescence resonance energy transfer-based kinase activity reporter for PKD to NHERF scaffolds reveals a unique signature of PKD activation at the scaffold that is distinct from that of general cytosolic or plasma membrane activity. Specifically, agonist-evoked activation of PKD at the scaffold is rapid and sustained but blunted in magnitude when compared with cytosolic PKD. Thus, live cell imaging of PKD activity demonstrates ultrasensitive control of kinase signaling at the scaffold compared with bulk activity in the cytosol or at the plasma membrane. Protein kinase D (PKD)2 plays a role in numerous processes including cell proliferation, cell survival, immune cell signaling, gene expression, vesicle trafficking, and neuronal development (1). The PKD family consists of three members belonging to the Ca 2ϩ /calmodulin-dependent kinase group of serine/threonine protein kinases. Each isoform contains a conserved catalytic core and an amino-terminal regulatory moiety. This regulatory region contains two cysteine-rich (C1) domains and a pleckstrin homology domain that autoinhibits the kinase (2). The C1 domains are membrane-targeting modules that bind diacylglycerol (DAG) and its functional analogues, phorbol esters, thus recruiting PKD to membranes (3). The PKD1 and PKD2 isoforms additionally contain PDZ-binding motifs at their carboxyl termini that can target the kinases to distinct subcellular scaffolds through interactions with PDZ domain-containing proteins (4).PKD transduces signals downstream of the second messenger DAG. In addition to membrane recruitment by DAG, activation of PKD requires phosphorylation by novel protein kinase C (PKC) family members at two sites within its catalytic core (5, 6). The novel PKCs themselves contain C1 domains and are allosterically activated by DAG-mediated membrane binding; thus, DAG production leads to PKD activation through coincident activation of the novel PKCs and localization of PKD near its upstream kinases. Hence, activation of phospholipase C (PLC)-coupled receptors (such as certain G protein-coupled receptors (GPCRs) or receptor tyrosine kinases) results in the production of second messengers including DA...

SummaryEnteropathogenic Escherichia coli induces characteristic attaching-effacing (A/E) lesions on the intestinal mucosa during infection. The locus of enterocyte effacement is essential for A/E lesion formation and encodes a type III secretion system that translocates multiple effector proteins into the host cell. Following translocation, EspI/NleA localizes to the Golgi. Using the yeast two-hybrid system (Y2HS) and PSD-95/Disklarge/ZO-1 (PDZ)-domain protein array overlays, we identified 15 putative host-interacting partners of EspI. All but two of the target proteins contained PDZ domains. Examination of the EspI amino acid sequence revealed a C-terminal consensus class I PDZ binding motif. Deletion of the last 7 amino acids of EspI to generate EspI DC7 abrogated the Y2HS interaction between EspI and 5 of the 6 putative host cell target proteins tested. Deletion of the EspI PDZ binding motif also resulted in delayed trafficking of EspI to the Golgi. Using a mouse model of infection, we showed that Citrobacter rodentium expressing truncated EspIDC7 was attenuated when in competition with C. rodentium expressing full-length EspI. Overall, these results suggested that EspI may modulate the virulence of A/E pathogens by binding host PDZ-domain proteins.

Our results demonstrate that ChK inhibits Akt kinase phosphorylation and cytokinesis in ras-transformed cells, which likely contribute to its ability to inhibit tumorigenic growth.