Adhesion G protein-coupled receptors: structure, signaling, physiology, and pathophysiology.

Adhesion G protein-coupled receptors (AGPCRs) are a family of 33 receptors in humans exhibiting a conserved general structure but diverse expression patterns and physiological functions. The large NH2 termini characteristic of AGPCRs confer unique properties to each receptor and possess a variety of distinct domains that can bind to a diverse array of extracellular proteins and components of the extracellular matrix. The traditional view of AGPCRs, as implied by their name, is that their core function is the mediation of adhesion. In recent years, though, many surprising advances have been made regarding AGPCR signaling mechanisms, activation by mechanosensory forces, and stimulation by small-molecule ligands such as steroid hormones and bioactive lipids. Thus, a new view of AGPCRs has begun to emerge in which these receptors are seen as massive signaling platforms that are crucial for the integration of adhesive, mechanosensory, and chemical stimuli. This review article describes the recent advances that have led to this new understanding of AGPCR function and also discusses new insights into the physiological actions of these receptors as well as their roles in human disease.

Mice lacking full length Adgrb1 (Bai1) exhibit social deficits, increased seizure susceptibility, and altered brain development.

The adhesion G protein-coupled receptor BAI1/ADGRB1 plays an important role in suppressing angiogenesis, mediating phagocytosis, and acting as a brain tumor suppressor. BAI1 is also a critical regulator of dendritic spine and excitatory synapse development and interacts with several autism-relevant proteins. However, little is known about the relationship between altered BAI1 function and clinically relevant phenotypes. Therefore, we studied the effect of reduced expression of full length Bai1 on behavior, seizure susceptibility, and brain morphology in Adgrb1 mutant mice. We compared homozygous (Adgrb1-/-), heterozygous (Adgrb1+/-), and wild-type (WT) littermates using a battery of tests to assess social behavior, anxiety, repetitive behavior, locomotor function, and seizure susceptibility. We found that Adgrb1-/- mice showed significant social behavior deficits and increased vulnerability to seizures. Adgrb1-/- mice also showed delayed growth and reduced brain weight. Furthermore, reduced neuron density and increased apoptosis during brain development were observed in the hippocampus of Adgrb1-/- mice, while levels of astrogliosis and microgliosis were comparable to WT littermates. These results show that reduced levels of full length Bai1 is associated with a broader range of clinically relevant phenotypes than previously reported.

The expanding functional roles and signaling mechanisms of adhesion G protein-coupled receptors.

The adhesion class of G protein-coupled receptors (GPCRs) is the second largest family of GPCRs (33 members in humans). Adhesion GPCRs (aGPCRs) are defined by a large extracellular N-terminal region that is linked to a C-terminal seven transmembrane (7TM) domain via a GPCR-autoproteolysis inducing (GAIN) domain containing a GPCR proteolytic site (GPS). Most aGPCRs undergo autoproteolysis at the GPS motif, but the cleaved fragments stay closely associated, with the N-terminal fragment (NTF) bound to the 7TM of the C-terminal fragment (CTF). The NTFs of most aGPCRs contain domains known to be involved in cell-cell adhesion, while the CTFs are involved in classical G protein signaling, as well as other intracellular signaling. In this workshop report, we review the most recent findings on the biology, signaling mechanisms, and physiological functions of aGPCRs.

A disease-associated mutation in the adhesion GPCR BAI2 (ADGRB2) increases receptor signaling activity.

Mutations in G protein-coupled receptors (GPCRs) that increase constitutive signaling activity can cause human disease. A de novo C-terminal mutation (R1465W) in the adhesion GPCR BAI2 (also known as ADGRB2) was identified in a patient suffering from progressive spastic paraparesis and other neurological symptoms. In vitro studies revealed that this mutation strongly increases the constitutive signaling activity of an N-terminally cleaved form of BAI2, which represents the activated form of the receptor. Further studies dissecting the mechanism(s) underling this effect revealed that wild-type BAI2 primarily couples to Gαz , with the R1465W mutation conferring increased coupling to Gαi . The R1465W mutation also increases the total and surface expression of BAI2. The mutation has no effect on receptor binding to ß-arrestins, but does perturb binding to the endocytic protein endophilin A1, identified here as a novel interacting partner for BAI2. These studies provide new insights into the signaling capabilities of the adhesion GPCR BAI2/ADGRB2 and shed light on how an apparent gain-of-function mutation to the receptor's C-terminus may lead to human disease.

Disease-associated extracellular loop mutations in the adhesion G protein-coupled receptor G1 (ADGRG1; GPR56) differentially regulate downstream signaling.

Mutations to the adhesion G protein-coupled receptor ADGRG1 (G1; also known as GPR56) underlie the neurological disorder bilateral frontoparietal polymicrogyria. Disease-associated mutations in G1 studied to date are believed to induce complete loss of receptor function through disruption of either receptor trafficking or signaling activity. Given that N-terminal truncation of G1 and other adhesion G protein-coupled receptors has been shown to significantly increase the receptors' constitutive signaling, we examined two different bilateral frontoparietal polymicrogyria-inducing extracellular loop mutations (R565W and L640R) in the context of both full-length and N-terminally truncated (ΔNT) G1. Interestingly, we found that these mutations reduced surface expression of full-length G1 but not G1-ΔNT in HEK-293 cells. Moreover, the mutations ablated receptor-mediated activation of serum response factor luciferase, a classic measure of Gα12/13-mediated signaling, but had no effect on G1-mediated signaling to nuclear factor of activated T cells (NFAT) luciferase. Given these differential signaling results, we sought to further elucidate the pathway by which G1 can activate NFAT luciferase. We found no evidence that ΔNT activation of NFAT is dependent on Gαq/11-mediated or ß-arrestin-mediated signaling but rather involves liberation of Gßγ subunits and activation of calcium channels. These findings reveal that disease-associated mutations to the extracellular loops of G1 differentially alter receptor trafficking, depending on the presence of the N terminus, and differentially alter signaling to distinct downstream pathways.

Group II metabotropic glutamate receptor interactions with NHERF scaffold proteins: Implications for receptor localization in brain.

The group II metabotropic glutamate receptors mGluR2 and mGluR3 are key modulators of glutamatergic neurotransmission. In order to identify novel Group II metabotropic glutamate receptor (mGluR)-interacting partners, we screened the C-termini of mGluR2 and mGluR3 for interactions with an array of PDZ domains. These screens identified the Na+/H+ exchanger regulatory factors 1 and 2 (NHERF-1 & -2) as candidate interacting partners. Follow-up co-immunoprecipitation studies demonstrated that both mGluR2 and mGluR3 can associate with NHERF-1 and NHERF-2 in a cellular context. Functional studies revealed that disruption of PDZ interactions with mGluR2 enhanced receptor signaling to Akt. However, further studies of mGluR2 and mGluR3 signaling in astrocytes in which NHERF expression was reduced by gene knockout (KO) and/or siRNA knockdown techniques revealed that the observed differences in signaling between WT and mutant mGluR2 were likely not due to disruption of interactions with the NHERF proteins. Electron microscopic analyses revealed that Group II mGluRs were primarily expressed in glia and unmyelinated axons in WT, NHERF-1 and NHERF-2 KO mice, but the relative proportion of labeled axons over glial processes was higher in NHERF-2 KO mice than in controls and NHERF-1 KO mice. Interestingly, our anatomical studies also revealed that loss of either NHERF protein results in ventriculomegaly, which may be related to the high incidence of hydrocephaly that has previously been observed in NHERF-1 KO mice. Together, these studies support a role for NHERF-1 and NHERF-2 in regulating the distribution of Group II mGluRs in the murine brain, while conversely the effects of the mGluR2/3 PDZ-binding motifs on receptor signaling are likely mediated by interactions with other PDZ scaffold proteins beyond the NHERF proteins.

PTEN recruitment controls synaptic and cognitive function in Alzheimer's models.

Dyshomeostasis of amyloid-ß peptide (Aß) is responsible for synaptic malfunctions leading to cognitive deficits ranging from mild impairment to full-blown dementia in Alzheimer's disease. Aß appears to skew synaptic plasticity events toward depression. We found that inhibition of PTEN, a lipid phosphatase that is essential to long-term depression, rescued normal synaptic function and cognition in cellular and animal models of Alzheimer's disease. Conversely, transgenic mice that overexpressed PTEN displayed synaptic depression that mimicked and occluded Aß-induced depression. Mechanistically, Aß triggers a PDZ-dependent recruitment of PTEN into the postsynaptic compartment. Using a PTEN knock-in mouse lacking the PDZ motif, and a cell-permeable interfering peptide, we found that this mechanism is crucial for Aß-induced synaptic toxicity and cognitive dysfunction. Our results provide fundamental information on the molecular mechanisms of Aß-induced synaptic malfunction and may offer new mechanism-based therapeutic targets to counteract downstream Aß signaling.

BAI1 regulates spatial learning and synaptic plasticity in the hippocampus.

Synaptic plasticity is the ability of synapses to modulate the strength of neuronal connections; however, the molecular factors that regulate this feature are incompletely understood. Here, we demonstrated that mice lacking brain-specific angiogenesis inhibitor 1 (BAI1) have severe deficits in hippocampus-dependent spatial learning and memory that are accompanied by enhanced long-term potentiation (LTP), impaired long-term depression (LTD), and a thinning of the postsynaptic density (PSD) at hippocampal synapses. We showed that compared with WT animals, mice lacking Bai1 exhibit reduced protein levels of the canonical PSD component PSD-95 in the brain, which stems from protein destabilization. We determined that BAI1 prevents PSD-95 polyubiquitination and degradation through an interaction with murine double minute 2 (MDM2), the E3 ubiquitin ligase that regulates PSD-95 stability. Restoration of PSD-95 expression in hippocampal neurons in BAI1-deficient mice by viral gene therapy was sufficient to compensate for Bai1 loss and rescued deficits in synaptic plasticity. Together, our results reveal that interaction of BAI1 with MDM2 in the brain modulates PSD-95 levels and thereby regulates synaptic plasticity. Moreover, these results suggest that targeting this pathway has therapeutic potential for a variety of neurological disorders.

International Union of Basic and Clinical Pharmacology. XCIV. Adhesion G protein-coupled receptors.

The Adhesion family forms a large branch of the pharmacologically important superfamily of G protein-coupled receptors (GPCRs). As Adhesion GPCRs increasingly receive attention from a wide spectrum of biomedical fields, the Adhesion GPCR Consortium, together with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification, proposes a unified nomenclature for Adhesion GPCRs. The new names have ADGR as common dominator followed by a letter and a number to denote each subfamily and subtype, respectively. The new names, with old and alternative names within parentheses, are: ADGRA1 (GPR123), ADGRA2 (GPR124), ADGRA3 (GPR125), ADGRB1 (BAI1), ADGRB2 (BAI2), ADGRB3 (BAI3), ADGRC1 (CELSR1), ADGRC2 (CELSR2), ADGRC3 (CELSR3), ADGRD1 (GPR133), ADGRD2 (GPR144), ADGRE1 (EMR1, F4/80), ADGRE2 (EMR2), ADGRE3 (EMR3), ADGRE4 (EMR4), ADGRE5 (CD97), ADGRF1 (GPR110), ADGRF2 (GPR111), ADGRF3 (GPR113), ADGRF4 (GPR115), ADGRF5 (GPR116, Ig-Hepta), ADGRG1 (GPR56), ADGRG2 (GPR64, HE6), ADGRG3 (GPR97), ADGRG4 (GPR112), ADGRG5 (GPR114), ADGRG6 (GPR126), ADGRG7 (GPR128), ADGRL1 (latrophilin-1, CIRL-1, CL1), ADGRL2 (latrophilin-2, CIRL-2, CL2), ADGRL3 (latrophilin-3, CIRL-3, CL3), ADGRL4 (ELTD1, ETL), and ADGRV1 (VLGR1, GPR98). This review covers all major biologic aspects of Adhesion GPCRs, including evolutionary origins, interaction partners, signaling, expression, physiologic functions, and therapeutic potential.

New functions and signaling mechanisms for the class of adhesion G protein-coupled receptors.

The class of adhesion G protein-coupled receptors (aGPCRs), with 33 human homologs, is the second largest family of GPCRs. In addition to a seven-transmembrane α-helix-a structural feature of all GPCRs-the class of aGPCRs is characterized by the presence of a large N-terminal extracellular region. In addition, all aGPCRs but one (GPR123) contain a GPCR autoproteolysis-inducing (GAIN) domain that mediates autoproteolytic cleavage at the GPCR autoproteolysis site motif to generate N- and a C-terminal fragments (NTF and CTF, respectively) during protein maturation. Subsequently, the NTF and CTF are associated noncovalently as a heterodimer at the plasma membrane. While the biological function of the GAIN domain-mediated autocleavage is not fully understood, mounting evidence suggests that the NTF and CTF possess distinct biological activities in addition to their function as a receptor unit. We discuss recent advances in understanding the biological functions, signaling mechanisms, and disease associations of the aGPCRs.

The BAI subfamily of adhesion GPCRs: synaptic regulation and beyond.

The brain-specific angiogenesis inhibitors 1-3 (BAI1-3) comprise a subfamily of adhesion G-protein-coupled receptors (GPCRs). These receptors are highly expressed in the brain and were first studied for their ability to inhibit angiogenesis and tumor formation. Subsequently, BAI1 was found to play roles in apoptotic cell phagocytosis and myoblast fusion. Until recently, however, little was known about the physiological importance of the BAI subfamily in the context of normal brain function. Recent work has provided evidence for key roles of BAI1-3 in the regulation of synaptogenesis and dendritic spine formation. In this review, we summarize the current understanding of the BAI subfamily with regard to downstream signaling pathways, physiological actions, and potential importance as novel drug targets in the treatment of psychiatric and neurological diseases.

JAM-A associates with ZO-2, afadin, and PDZ-GEF1 to activate Rap2c and regulate epithelial barrier function.

Intestinal barrier function is regulated by epithelial tight junctions (TJs), structures that control paracellular permeability. Junctional adhesion molecule-A (JAM-A) is a TJ-associated protein that regulates barrier; however, mechanisms linking JAM-A to epithelial permeability are poorly understood. Here we report that JAM-A associates directly with ZO-2 and indirectly with afadin, and this complex, along with PDZ-GEF1, activates the small GTPase Rap2c. Supporting a functional link, small interfering RNA-mediated down-regulation of the foregoing regulatory proteins results in enhanced permeability similar to that observed after JAM-A loss. JAM-A-deficient mice and cultured epithelial cells demonstrate enhanced paracellular permeability to large molecules, revealing a potential role of JAM-A in controlling perijunctional actin cytoskeleton in addition to its previously reported role in regulating claudin proteins and small-molecule permeability. Further experiments suggest that JAM-A does not regulate actin turnover but modulates activity of RhoA and phosphorylation of nonmuscle myosin, both implicated in actomyosin contraction. These results suggest that JAM-A regulates epithelial permeability via association with ZO-2, afadin, and PDZ-GEF1 to activate Rap2c and control contraction of the apical cytoskeleton.

Brain-specific angiogenesis inhibitor-1 signaling, regulation, and enrichment in the postsynaptic density.

Brain-specific angiogenesis inhibitor-1 (BAI1) is an adhesion G protein-coupled receptor that has been studied primarily for its anti-angiogenic and anti-tumorigenic properties. We found that overexpression of BAI1 results in activation of the Rho pathway via a Gα(12/13)-dependent mechanism, with truncation of the BAI1 N terminus resulting in a dramatic enhancement in receptor signaling. This constitutive activity of the truncated BAI1 mutant also resulted in enhanced downstream phosphorylation of ERK as well as increased receptor association with ß-arrestin2 and increased ubiquitination of the receptor. To gain insights into the regulation of BAI1 signaling, we screened the C terminus of BAI1 against a proteomic array of PDZ domains to identify novel interacting partners. These screens revealed that the BAI1 C terminus interacts with a variety of PDZ domains from synaptic proteins, including MAGI-3. Removal of the BAI1 PDZ-binding motif resulted in attenuation of receptor signaling to Rho but had no effect on ERK activation. Conversely, co-expression with MAGI-3 was found to potentiate signaling to ERK by constitutively active BAI1 in a manner that was dependent on the PDZ-binding motif of the receptor. Biochemical fractionation studies revealed that BAI1 is highly enriched in post-synaptic density fractions, a finding consistent with our observations that BAI1 can interact with PDZ proteins known to be concentrated in the post-synaptic density. These findings demonstrate that BAI1 is a synaptic receptor that can activate both the Rho and ERK pathways, with the N-terminal and C-terminal regions of the receptor playing key roles in the regulation of BAI1 signaling activity.

GPR37 and GPR37L1 are receptors for the neuroprotective and glioprotective factors prosaptide and prosaposin.

GPR37 (also known as Pael-R) and GPR37L1 are orphan G protein-coupled receptors that are almost exclusively expressed in the nervous system. We screened these receptors for potential activation by various orphan neuropeptides, and these screens yielded a single positive hit: prosaptide, which promoted the endocytosis of GPR37 and GPR37L1, bound to both receptors and activated signaling in a GPR37- and GPR37L1-dependent manner. Prosaptide stimulation of cells transfected with GPR37 or GPR37L1 induced the phosphorylation of ERK in a pertussis toxin-sensitive manner, stimulated (35)S-GTPγS binding, and promoted the inhibition of forskolin-stimulated cAMP production. Because prosaptide is the active fragment of the secreted neuroprotective and glioprotective factor prosaposin (also known as sulfated glycoprotein-1), we purified full-length prosaposin and found that it also stimulated GPR37 and GPR37L1 signaling. Moreover, both prosaptide and prosaposin were found to protect primary astrocytes against oxidative stress, with these protective effects being attenuated by siRNA-mediated knockdown of endogenous astrocytic GPR37 or GPR37L1. These data reveal that GPR37 and GPR37L1 are receptors for the neuroprotective and glioprotective factors prosaptide and prosaposin.

SAP97 promotes the stability of Nax channels at the plasma membrane.

Na(x) is a sodium-level sensor for body fluids expressed in the circumventricular organs in the brain. Na(x) has a putative PSD-95/Disc-large/ZO-1 (PDZ)-binding motif at the carboxyl (C)-terminus. Here we found that several PDZ proteins bind to Na(x) by PDZ-array overlay assay. Among them, synapse-associated protein 97 (SAP97/DLG1) was coexpressed with Na(x) in the subfornical organ. In C6 glioblastoma cells, destruction of the PDZ-binding motif of Na(x) or depletion of SAP97 resulted in a decrease in cell-surface Na(x), which was attenuated with inhibitors of endocytosis. These results indicate that SAP97 contributes to the stabilization of Na(x) channels at the plasma membrane.

A new human NHERF1 mutation decreases renal phosphate transporter NPT2a expression by a PTH-independent mechanism.

BACKGROUND: The sodium-hydrogen exchanger regulatory factor 1 (NHERF1) binds to the main renal phosphate transporter NPT2a and to the parathyroid hormone (PTH) receptor. We have recently identified mutations in NHERF1 that decrease renal phosphate reabsorption by increasing PTH-induced cAMP production in the renal proximal tubule. METHODS: We compared relevant parameters of phosphate homeostasis in a patient with a previously undescribed mutation in NHERF1 and in control subjects. We expressed the mutant NHERF1 protein in Xenopus Oocytes and in cultured cells to study its effects on phosphate transport and PTH-induced cAMP production. RESULTS: We identified in a patient with inappropriate renal phosphate reabsorption a previously unidentified mutation (E68A) located in the PDZ1 domain of NHERF1.We report the consequences of this mutation on NHERF1 function. E68A mutation did not modify cAMP production in the patient. PTH-induced cAMP synthesis and PKC activity were not altered by E68A mutation in renal cells in culture. In contrast to wild-type NHERF1, expression of the E68A mutant in Xenopus oocytes and in human cells failed to increase phosphate transport. Pull down experiments showed that E68A mutant did not interact with NPT2a, which robustly interacted with wild type NHERF1 and previously identified mutants. Biotinylation studies revealed that E68A mutant was unable to increase cell surface expression of NPT2a. CONCLUSIONS: Our results indicate that the PDZ1 domain is critical for NHERF1-NPT2a interaction in humans and for the control of NPT2a expression at the plasma membrane. Thus we have identified a new mechanism of renal phosphate loss and shown that different mutations in NHERF1 can alter renal phosphate reabsorption via distinct mechanisms.

Adenosine A2A receptor in the monkey basal ganglia: ultrastructural localization and colocalization with the metabotropic glutamate receptor 5 in the striatum.

The adenosine A(2A) receptor (A(2A) R) is a potential drug target for the treatment of Parkinson's disease and other neurological disorders. In rodents, the therapeutic efficacy of A(2A) R modulation is improved by concomitant modulation of the metabotropic glutamate receptor 5 (mGluR5). To elucidate the anatomical substrate(s) through which these therapeutic benefits could be mediated, pre-embedding electron microscopy immunohistochemistry was used to conduct a detailed, quantitative ultrastructural analysis of A(2A) R localization in the primate basal ganglia and to assess the degree of A(2A) R/mGluR5 colocalization in the striatum. A(2A) R immunoreactivity was found at the highest levels in the striatum and external globus pallidus (GPe). However, the monkey, but not the rat, substantia nigra pars reticulata (SNr) also harbored a significant level of neuropil A(2A) R immunoreactivity. At the electron microscopic level, striatal A(2A) R labeling was most commonly localized in postsynaptic elements (58% ± 3% of labeled elements), whereas, in the GPe and SNr, the labeling was mainly presynaptic (71% ± 5%) or glial (27% ± 6%). In both striatal and pallidal structures, putative inhibitory and excitatory terminals displayed A(2A) R immunoreactivity. Striatal A(2A) R/mGluR5 colocalization was commonly found; 60-70% of A(2A) R-immunoreactive dendrites or spines in the monkey striatum coexpress mGluR5. These findings provide the first detailed account of the ultrastructural localization of A(2A) R in the primate basal ganglia and demonstrate that A(2A) R and mGluR5 are located to interact functionally in dendrites and spines of striatal neurons. Together, these data foster a deeper understanding of the substrates through which A(2A) R could regulate primate basal ganglia function and potentially mediate its therapeutic effects in parkinsonism.

Protein kinase Cα promotes cell migration through a PDZ-dependent interaction with its novel substrate discs large homolog 1 (DLG1).

Protein scaffolds maintain precision in kinase signaling by coordinating kinases with components of specific signaling pathways. Such spatial segregation is particularly important in allowing specificity of signaling mediated by the 10-member family of protein kinase C (PKC) isozymes. Here we identified a novel interaction between PKCα and the Discs large homolog (DLG) family of scaffolds that is mediated by a class I C-terminal PDZ (PSD-95, disheveled, and ZO1) ligand unique to this PKC isozyme. Specifically, use of a proteomic array containing 96 purified PDZ domains identified the third PDZ domains of DLG1/SAP97 and DLG4/PSD95 as interaction partners for the PDZ binding motif of PKCα. Co-immunoprecipitation experiments verified that PKCα and DLG1 interact in cells by a mechanism dependent on an intact PDZ ligand. Functional assays revealed that the interaction of PKCα with DLG1 promotes wound healing; scratch assays using cells depleted of PKCα and/or DLG1 have impaired cellular migration that is no longer sensitive to PKC inhibition, and the ability of exogenous PKCα to rescue cellular migration is dependent on the presence of its PDZ ligand. Furthermore, we identified Thr-656 as a novel phosphorylation site in the SH3-Hook region of DLG1 that acts as a marker for PKCα activity at this scaffold. Increased phosphorylation of Thr-656 is correlated with increased invasiveness in non-small cell lung cancer lines from the NCI-60, consistent with this phosphorylation site serving as a marker of PKCα-mediated invasion. Taken together, these data establish the requirement of scaffolding to DLG1 for PKCα to promote cellular migration.

CART peptide stimulation of G protein-mediated signaling in differentiated PC12 cells: identification of PACAP 6-38 as a CART receptor antagonist.

CART peptides are peptide neurotransmitters and hormones that are involved in many different physiological responses. While much is known about the peptides regarding their structure, processing and gene regulation, less is known about their postsynaptic actions and receptors. Using (125)I-CART 61-102 as a ligand and unlabeled CART 61-102 or CART 55-102 as displacers, high-affinity specific binding was detected in PC12 cells. Differentiation of the PC12 cells increased specific binding several-fold. The increase in specific binding found after differentiation was inhibited by actinomycin D and cycloheximide, suggesting that the increase in specific binding was dependent on RNA and protein synthesis. CART 1-27, a peptide that has never been shown to elicit responses, did not displace (125)I-CART 61-102 binding, nor did more than 20 other peptides that were examined. Surprisingly, however, PACAP 1-38 and PACAP 6-38 were found to be low-affinity inhibitors of CART binding. CART treatment increased binding of (35)S-GTPgamma-S to PC12 cell membranes. Moreover, CART treatment of intact PC12 cells elicited robust increases in phospho-ERK in a manner that was increased with differentiation, blocked by pertussis toxin and antagonized by PACAP 6-38. These findings extend previous research and suggest that the CART binding site in PC12 cells reflects a G protein-coupled receptor linked with Gi/o, and also demonstrate that PACAP 6-38 may be useful as a CART receptor antagonist.

MAGI-3 competes with NHERF-2 to negatively regulate LPA2 receptor signaling in colon cancer cells.

BACKGROUND & AIMS: Lysophosphatidic acid (LPA) is a potent inducer of colon cancer and LPA receptor type 2 (LPA(2)) is overexpressed in colon tumors. LPA(2) interacts with membrane-associated guanylate kinase with inverted orientation-3 (MAGI-3) and the Na+/H+ exchanger regulatory factor 2 (NHERF-2), but the biological effects of these interactions are unknown. We investigated the roles of MAGI-3 and NHERF-2 in LPA(2)-mediated signaling in human colon cancer cells. METHODS: We overexpressed or knocked down MAGI-3 in HCT116 and SW480 cells. The effects of MAGI-3 and NHERF-2 in LPA-induced cell migration, invasion, inositol phosphate generation, and nuclear factor-κB activation were determined. Expression of MAGI-3 and NHERF-2 in human colon tumor tissues was analyzed using tissue microarray analysis. RESULTS: NHERF-2 promoted migration and invasion of colon cancer cells, whereas MAGI-3 inhibited these processes. MAGI-3 competed with NHERF-2 for binding to LPA(2) and phospholipase C-ß3. However, NHERF-2 and MAGI-3 reciprocally regulated LPA(2)-induced phospholipase C activity. MAGI-3 increased the interaction of LPA(2) with Gα(12), whereas NHERF-2 preferentially promoted interaction between LPA(2) and Gα(q). MAGI-3 decreased the tumorigenic capacity of LPA(2) by attenuating the activities of nuclear factor-κB and c-Jun N-terminal kinase. MAGI-3 and NHERF-2 were expressed differentially in colon adenocarcinomas, consistent with their opposing effects. CONCLUSIONS: LPA(2) is dynamically regulated by 2 distinct PDZ proteins via modulation of G-protein coupling and receptor signaling. MAGI-3 is a negative regulator of LPA(2) signaling.