Calmodulin (CaM) binds in a Ca2+-dependent manner to the intracellular C-terminal domains of most group III metabotropic glutamate receptors (mGluRs). Here
we combined mutational and biophysical approaches to define the structural basis of CaM binding to mGluR 7A. Ca2+/CaM was found to interact with mGluR 7A primarily via its C-lobe at a 1:1 CaM:C-tail stoichiometry. Pulldown experiments
with mutant CaM and mGluR 7A C-tail constructs and high resolution NMR with peptides corresponding to the CaM binding region
of mGluR 7A allowed us to define hydrophobic and ionic interactions required for Ca2+/CaM binding and identified a 1-8-14 CaM-binding motif. The Ca2+/CaM·mGluR 7A peptide complex displays a classical wraparound structure that closely resembles that formed by Ca2+/CaM upon binding to smooth muscle myosin light chain kinase. Our data provide insight into how Ca2+/CaM regulates group III mGluR signaling via competition with intracellular proteins for receptor-binding sites.
"Structural studies indicate that Ca 2+ /CaM binding to mGlu7a includes interactions with both Ser 862 , a target site for phosphorylation by protein kinase C (Nakajima et al., 1999; Dev et al., 2000; Airas et al., 2001), and the G bg recognition motif (El Far et al., 2001). Recent structural studies have also identified the region from amino acids 856–879 of mGlu7 C-tail to be responsible for CaM binding (Scheschonka et al., 2008). Moreover, the C-tail of mGlu7a displays a PDZ-binding motif (LVI) that has been shown to interact with PICK1 (protein interacting with C kinase 1) (El Far et al., 2000), and this interaction is involved in synaptic transmission and plasticity (Perroy et al., 2002). "
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND AND PURPOSE The mGlu7 receptors are strategically located at the site of vesicle fusion where they modulate the release of the main excitatory and inhibitory neurotransmitters. Consequently, they are implicated in the underlying pathophysiology of CNS diseases such as epilepsy and stress-related psychiatric disorders. Here, we characterized a selective, potent and functional anti-mGlu7 monoclonal antibody, MAB1/28, that triggers receptor internalization.
EXPERIMENTAL APPROACH MAB1/28's activity was investigated using Western blot and direct immunofluorescence on live cells, in vitro pharmacology by functional cAMP and [35S]-GTPγ binding assays, the kinetics of IgG-induced internalization by image analysis, and the activation of the ERK1/2 by elisa.
KEY RESULTS mGlu7/mGlu6 chimeric studies located the MAB1/28 binding site at the extracellular amino-terminus of mGlu7. MAB1/28 potently antagonized both orthosteric and allosteric agonist-induced inhibition of cAMP accumulation. The potency of the antagonistic actions was similar to the potency in triggering receptor internalization. The internalization mechanism occurred via a pertussis toxin-insensitive pathway and did not require Gαi protein activation. MAB1/28 activated ERK1/2 with potency similar to that for receptor internalization. The requirement of a bivalent receptor binding mode for receptor internalizations suggests that MAB1/28 modulates mGlu7 dimers.
CONCLUSIONS AND IMPLICATIONS We obtained evidence for an allosteric-biased agonist activity triggered by MAB1/28, which activates a novel IgG-mediated GPCR internalization pathway that is not utilized by small molecule, orthosteric or allosteric agonists. Thus, MAB1/28 provides an invaluable biological tool for probing mGlu7 function and selective activation of its intracellular trafficking.
British Journal of Pharmacology 07/2012; 167(7). DOI:10.1111/j.1476-5381.2012.02090.x · 4.84 Impact Factor
"proteins. This hypothesis is supported by experimental data: The mGluR7a-CT forms an amphipathic a-helix in contact with Calmodulin , while 5 amino acids of the mGluR7b-CT adopt an extended b-sheet conformation in complex with PP1  . "
[Show abstract][Hide abstract] ABSTRACT: Metabotropic glutamate receptors (mGluRs) are regulated by interacting proteins that mostly bind to their intracellular C-termini. Here, we investigated if mGluR6, mGluR7a and mGluR8a C-termini form predefined binding surfaces or if they were rather unstructured. Limited tryptic digest of purified peptides argued against the formation of stable globular folds. Circular dichroism, (1)H NMR and (1)H(15)N HSQC spectra indicated the absence of rigid secondary structure elements. Furthermore, we localized short linear binding motifs in the unstructured receptor domains. Our data provide evidence that protein interactions of the analyzed mGluR C-termini are mediated rather by short linear motifs than by preformed folds.
[Show abstract][Hide abstract] ABSTRACT: The hardware/software partitioning/scheduling relies on two subtasks: the cost function and the real time (RT) analysis. Besides these two subtasks, the proposed generic framework, also called RT design trotter (RTDT), processes the problem of the Quality of Service (QoS) management. The aim is to add a new dimensions to solution selection, namely the guarantee of QoS from both application quality and RT issue points of view. The proposed framework defines an iteration loop of three steps that solve the sub-problems. The cost function takes into account the system on chip (SoC) area and the static and dynamic power dissipation. We show how our tool can be used to rapidly evaluate the impact of the application quality and the RT constraints choices (QoS parameters) over the final cost.
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.