Venus Fly Trap Domain of mGluR1 Functions as a Dominant Negative Against Group I mGluR Signaling

Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York 14642, USA.
Journal of Neurophysiology (Impact Factor: 2.89). 05/2010; 104(1):439-48. DOI: 10.1152/jn.00799.2009
Source: PubMed


Metabotropic glutamate receptors (mGluRs) form covalently linked homodimers and contain large, N-terminal extracellular ligand binding, "venus fly trap" (VFT) domains. These domains, when expressed separately, are secreted as disulfide linked dimers and can dimerize with full-length receptors. mGluR splice variants have been described that contain only this domain, but the consequences of their interaction on receptor signaling have not been explored. Here it is shown that an mGluR1 mutant containing only the VFT is retained on the cell surface when a full-length receptor is co-expressed. Further, when expressed in rat superior cervical ganglion (SCG) neurons and modulation of native calcium currents is used as an assay for receptor activity, the VFT acts as a dominant negative with respect to mGluR1 signaling. Although full-length mGluR1 and mGluR5 are not known to heterodimerize, the mGluR5 VFT partially occludes mGluR1 signaling and the mGluR1 VFT potently occludes mGluR5 signaling in SCG neurons. In addition, an mGluR1 point mutant, mGluR1 C140G, which cannot covalently dimerize, functions like the wild-type receptor when expressed alone. The C140G mutant is inhibited by the mGluR1 VFT construct but does not retain the mGluR1 VFT on the cell surface, suggesting that the loss of C140 renders the interaction reversible. Finally, a peptide designed to disrupt mGluR1 dimerization reduced signaling through the C140G mutant receptor, but only when applied intracellularly for several hours, indicating that loss of signaling requires disruption of dimerization prior to plasma membrane insertion.

9 Reads
  • Source
    • "Responses peaked at nearly 80% inhibition with an apparent EC 50 of about 56 mM. Cells expressing mGluR1 alone had responses as expected, peaking at approximately 40% inhibition and with an apparent EC 50 of about 1 mM, consistent with previous reports (Kammermeier and Yun, 2005; Beqollari and Kammermeier, 2010). As with the mGluR1 Y74A/mGluR5 combination, when mGluR5 Y64A was coexpressed with mGluR1, responses showed strong efficacy and intermediate potency (EC 50 of 16 mM) compared with each receptor alone. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Metabotropic glutamate receptors (mGluRs) function as dimers. Recent work suggests that mGluR1 and mGluR5 may physically interact, but the nature and functional consequences of this relationship have not been addressed. In this study, the functional and pharmacological consequences of this interaction were investigated. Using heterologous expression of mGluR cDNA in rat sympathetic neurons from the superior cervical ganglion (SCG) and inhibition of the native calcium currents as an assay for receptor activation, a functional interdependence between mGluR1 and 5 was demonstrated. In neurons co-expressing these receptors, combining a selective mGluR1 competitive antagonist with either an mGluR1 or mGluR5 selective negative allosteric modulator (NAM: BAY36 7620 and MPEP, respectively) strongly occluded signaling by both receptors to an approximately equal degree. By contrast, in cells co-expressing mGluR1 and 2, combining the same mGluR1 competitive inhibitor with an mGluR1 or mGluR2 NAM yielded partial and full inhibition of the response, respectively, as expected for independently acting receptors. In neurons expressing mGluR1 and 5, the selective NAMs each strongly inhibited the response to glutamate, suggesting that these receptors do not interact as heterodimers, which would not be inhibited by selective NAMs. Finally, evidence for a similar mGluR1/5 functional dependence is shown in medium spiny striatal neurons. Together, these data demonstrate cooperative signaling between mGluR1 and 5 in a manner inconsistent with heterodimerization, and thus suggesting an interaction between homodimers.
    Molecular pharmacology 08/2014; 86(5). DOI:10.1124/mol.114.093468 · 4.13 Impact Factor
  • Source
    • "All mGluRs share a basic architecture (Figure 4 and 5). At the N-terminus exists a large bi-lobed extracellular domain known as the amino terminal domain (ATD), which in the protein crystallography literature is also referred to as the venus fly trap domain (VFT) because of its unique shape 16. Following the ATD is a cysteine-rich domain (CRD) which is critical for dimerization and activation 17. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Glutamate is a nonessential amino acid, a major bioenergetic substrate for proliferating normal and neoplastic cells, and an excitatory neurotransmitter that is actively involved in biosynthetic, bioenergetic, metabolic, and oncogenic signaling pathways. Glutamate signaling activates a family of receptors consisting of metabotropic glutamate receptors (mGluRs) and ionotropic glutamate receptors (iGluRs), both of which have been implicated in chronic disabling brain disorders such as Schizophrenia and neurodegenerative diseases like Alzheimer's, Parkinson's, and multiple sclerosis. In this review, we discuss the structural and functional relationship of mGluRs and iGluRs and their downstream signaling pathways. The three groups of mGluRs, the associated second messenger systems, and subsequent activation of PI3K/Akt, MAPK, NFkB, PLC, and Ca/CaM signaling systems will be discussed in detail. The current state of human mGluR1a as one of the most important isoforms of Group I-mGluRs will be highlighted. The lack of studies on the human orthologues of mGluRs family will be outlined. We conclude that upon further study, human glutamate-initiated signaling pathways may provide novel therapeutic opportunities for a variety of non-malignant and malignant human diseases.
    International journal of biological sciences 09/2013; 9(9):948-959. DOI:10.7150/ijbs.6426 · 4.51 Impact Factor
  • Source
    • "First, the ATD is far larger than other GPCR family members at approximately 55 kDa 8. The unique two-lobed shape of this domain in which Glu binds between the two lobes is referred to as the “venus fly trap” domain 9. Second, the receptors differ from classic GPCRs and further the isoforms differ from one another at the level of the CTD. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Glutamate, a nonessential amino acid, is the major excitatory neurotransmitter in the central nervous system. As such, glutamate has been shown to play a role in not only neural processes, such as learning and memory, but also in bioenergetics, biosynthetic and metabolic oncogenic pathways. Glutamate has been the target of intense investigation for its involvement not only in the pathogenesis of benign neurodegenerative diseases (NDDs) such as Parkinson's disease, Alzheimer's disease, schizophrenia, multiple sclerosis, and amyotropic lateral sclerosis (ALS), but also in carcinogenesis and progression of malignant diseases. In addition to its intracellular activities, glutamate in secreted form is a phylogenetically conserved cell signaling molecule. Glutamate binding activates multiple major receptor families including the metabotropic glutamate receptors (mGluRs) and ionotropic glutamate receptors (iGluRs), both of which have been implicated in various signaling pathways in cancer. Inhibition of extracellular glutamate release or glutamate receptor activation via competitive or non-competitive antagonists decreases growth, migration and invasion and induces apoptosis in breast cancer, melanoma, glioma and prostate cancer cells. In this review, we discuss the current state of glutamate signaling research as it relates to benign and malignant diseases. In addition, we provide a synopsis of clinical trials using glutamate antagonists for the treatment of NDD and malignant diseases. We conclude that in addition to its potential role as a metabolic biomarker, glutamate receptors and glutamate-initiated signaling pathways may provide novel therapeutic opportunities for cancer.
    International journal of biological sciences 08/2013; 9(7):728-42. DOI:10.7150/ijbs.6475 · 4.51 Impact Factor
Show more

Similar Publications