Francisco Ciruela

Metabotropic Glutamate 1α and Adenosine A1 Receptors Assemble into Functionally Interacting Complexes

Recently, evidence has emerged that seven transmembrane G protein-coupled receptors may be present as homo- and heteromers in the plasma membrane. Here we describe a new molecular and functional interaction between two functionally unrelated types of G protein-coupled receptors, namely the metabotropic glutamate type 1α (mGlu1α receptor) and the adenosine A1 receptors in cerebellum, primary cortical neurons, and heterologous transfected cells. Co-immunoprecipitation experiments showed a close and subtype-specific interaction between mGlu1α and A1 receptors in both rat cerebellar synaptosomes and co-transfected HEK-293 cells. By using transiently transfected HEK-293 cells a synergy between mGlu1α and A1 receptors in receptor-evoked [Ca2+] i signaling has been shown. In primary cultures of cortical neurons we observed a high degree of co-localization of the two receptors, and excitotoxicity experiments in these cultures also indicate that mGlu1α and A1 receptors are functionally related. Our results provide a molecular basis for adenosine/glutamate receptors cross-talk and open new perspectives for the development of novel agents to treat neuropsychiatric disorders in which abnormal glutamatergic neurotransmission is involved.

Homer-1c/Vesl-1L Modulates the Cell Surface Targeting of Metabotropic Glutamate Receptor Type 1α: Evidence for an Anchoring Function

Homer-1c/Vesl-1L is a 48-kDa protein that forms part of a family of conserved Homer-related proteins that interact with the C-termini of the metabotropic glutamate receptors mGluR1alpha and mGluR5. In order to examine the function of Homer-1c, HEK-293 cells have been transfected with mGluR1alpha, Homer-1c, and both proteins together. When cells were transfected with both proteins, biotinylation of cell surface molecules revealed a significant increase in the amount of receptor and Homer-1c associated with the cell surface compared with cells transfected with mGluR1alpha alone. This finding was paralleled by a concomitant increase in the production of inositol after treatment of the doubly transfected cells with agonist. Cell surface immunostaining of mGluR1alpha showed that Homer-1c can induce clustering of the receptor in the plasma membrane of HEK-293 cells and suggested that the surface receptor was associated with Homer-1c in the plasma membrane. The presence of Homer-1c reduced the rate of loss from the cell surface of mGluR1alpha from 5 to 1%/min and increased the extent of dendritic trafficking of the receptor in rat primary cultured neurons. Our results suggest that Homer-1c increases the cell surface expression of the metabotropic glutamate receptor type 1alpha by increasing its retention in the plasma membrane.

Characterization of the Dimerization of Metabotropic Glutamate Receptors Using an N‐Terminal Truncation of mGluR1α

Abstract: The metabotropic glutamate receptor mGluR1α in membranes isolated both from rat brain and from cell lines transfected with cDNA coding for the receptor migrates as a disulphide‐bonded dimer on sodium dodecyl sulphate‐polyacrylamide gels. Dimerization of mGluR1α takes place in the endoplasmic reticulum because it is not prevented by exposing transfected human embryonic kidney (HEK) 293 cells to the drug brefeldin A, a drug that prevents egress of proteins from the endoplasmic reticulum. Dimerization was also not dependent on protein glycosylation as it was not prevented by treatment of the cells with tunicamycin. Using a mammalian expression vector containing the N‐terminal domain of mGluR1α, truncated just before the first transmembrane domain (NT‐mGluR1α), we show that the N‐terminal domain is secreted as a soluble disulphide‐bonded dimeric protein. In addition, the truncated N‐terminal domain can form heterodimers with mGluR1α when both proteins are cotransfected into HEK 293 cells. However, mGluR1α and its splice variant mGluR1β did not form heterodimers in doubly transfected HEK 293 cells. These results show that although the N‐terminal domain of mGluR1α is sufficient for dimer formation, other domains in the molecule must regulate the process.

Differential internalisation of mGluR1 splice variants in response to agonist and phorbol esters in permanently transfected BHK cells

The internalisation of metabotropic glutamate receptor (mGluR1α) and its splice variant (mGluR1β), in response to agonist and phorbol esters (PMA), has been studied. Both mGluR1α and mGluR1β exhibit a similar rate of internalisation following PMA treatment, with a shift in their distribution from plasma membrane to endosome‐enriched membrane fractions. Agonist challenge however caused a rapid loss, within 5–10 min, of mGluR1β but not mGluR1α from the cell surface. These results show that the two forms of mGluR1 show different internalisation responses to agonist and suggest that the C‐terminal region of the molecule plays an important role in this phenomenon.

Interactions of the C terminus of Metabotropic glutamate receptor type 1α with rat brain proteins : Evidence for a direct interaction with tubulin

Abstract : Metabotropic glutamate receptors (mGluRs) are coupled to G protein second messenger pathways and modulate glutamate neurotransmission in the brain, where they are targeted to specific synaptic locations. As part of a strategy for defining the mechanisms for the specific targeting of mGluR1 α, rat brain proteins which interact with the intracellular carboxy terminus of mGluR1 α have been characterized, using affinity chromatography on a glutathione S‐transferase fusion protein that contains the last 86 amino acids of mGluR1 α. Three of the proteins specifically eluted from the affinity column yielded protein sequences, two of which were identified as glyceraldehyde‐3‐phosphate dehydrogenase and β‐tubulin ; the other was an unknown protein. The identity of tubulin was confirmed by western immunoblotting. Using a solid‐phase binding assay, the mGluR1 α‐tubulin interaction was shown to be direct, specific, and saturable with a KD of 2.3 ± 0.4 μM. In addition, mGluR1 α, but not mGluR2/3 or mGluR4, could be coimmunoprecipitated from solubilized brain extracts with tubulin using anti‐β‐tubulin antibodies. However, mGluR1 α could not be coimmunoprecipitated with the tubulin binding protein gephyrin, nor could it be coimmunoprecipitated with PSD95. Collectively these data demonstrate that the last 86 amino acids of the carboxyl‐terminal tail of mGluR1 α are sufficient to determine its interaction with tubulin and that there is an association of this receptor with tubulin in rat brain.

Molecular determinants of metabotropic glutamate receptor 1B trafficking.

The metabotropic glutamate receptor mGluR1 undergoes alternative splicing to generate isoforms differing in C-terminal sequence. The mechanism by which these isoforms give different functional responses to agonists in vitro is so far unclear. Using the native mGluR1 and CD2-mGluR1 chimeric molecules, as well as their C-terminal truncations and mutants, we identified an endoplasmic reticulum (ER) retention signal Arg-Arg-Lys-Lys within the C-terminal sequence of mGluR1b. Its presence results in a much reduced cell surface expression of the receptor and chimeric molecules in cell lines and their restricted trafficking in neurones. This motif is also present in the C-terminus of mGluR1a, but its effect is overcome by a region of the mGluR1a-specific C-terminal sequence (amino acids 975-1098). Our results indicate that these splice variants of mGluR1 utilize different targeting pathways and suggest that this may be a general phenomenon in the metabotropic glutamate receptor gene family.

Metabotropic glutamate receptor type 1α and tubulin assemble into dynamic interacting complexes

Metabotropic glutamate receptors (mGlu receptors) are coupled to G‐protein second messenger pathways and modulate glutamate neurotransmission in the brain, where they are targeted to specific synaptic locations. Very recently, we identified tubulin as an interacting partner of the mGlu1α receptor in rat brain. Using BHK‐570 cells permanently expressing the receptor we have shown that this interaction occurs predominantly with soluble tubulin, following its translocation to the plasma membrane. In addition, treatment of the cells with the agonist quisqualic acid induce tubulin depolimerization and its translocation to the plasma membrane. Immunofluorescence detection of both the receptor and tubulin in agonist‐treated cells reveals a disruption of the microtubule network and an increased clustering of the receptor. Collectively these data demonstrate that the mGlu1α receptor interacts with soluble tubulin and that this association can take place at the plasma membrane.

Receptor-receptor interactions in heteroreceptor complexes: a new principle in biology. Focus on their role in learning and memory

The allosteric receptor-receptor interactions over the interfaces in heteroreceptor complexes have been explored and their biochemical, pharmacological and functional integrative implications in the Central Nervous System (CNS) described. GPCR interacting proteins participate in these complexes mainly through modulation of receptor-receptor interactions. Methodologies to study heteroreceptor complexes in living cells (FRET and BRET-based techniques) and in brain tissue (in situ proximity ligation assay) are briefly summarized. The physiological and pathological relevance of the allosteric receptor-receptor interactions in heteroreceptor complexes is emphasized and novel strategies for treatment of mental and neurological disease are developed based on this new biological principle of integration. The molecular basis of learning and memory is proposed to be based on the reorganization of the homo- and heteroreceptor complexes in the postjunctional membrane of synapses leading also to changes in the prejunctional receptor complexes to facilitate the pattern of transmitter release to be learned. Long-term memory may be created by the transformation of parts of the heteroreceptor complexes into unique transcription factors which can lead to the formation of specific adapter proteins which can consolidate the heteroreceptor complexes into long-lived complexes with conserved allosteric receptor-receptor interactions.

Cell surface expression of the metabotropic glutamate receptor type 1α is regulated by the C-terminal tail

The cell surface expression of metabotropic glutamate receptor type 1 splice variants has been studied using cell surface biotinylation. Co‐expression of the last 86 residues of the C‐terminal tail of mGluR1α (F2‐protein) with mGluR1α caused a significant reduction of the amount of the cell surface receptor when compared to that in cells transfected with mGlur1α alone, and this was accompanied by a reduction in the production of inositol following agonist stimulation of the cells. In contrast, cell surface expression of mGluR1β was unaltered by co‐expression with the F2‐protein. These results suggest that the C‐terminal tail of mGluR1α regulates cell surface expression of the receptor.

Identification, cloning and analysis of expression of a new alternatively spliced form of the metabotropic glutamate receptor mGluR1 mRNA

Abstract We have applied quantitative RT-PCR analysis to characterise relative levels of expression of the alternatively spliced mGluR1 mRNAs. This has also allowed us to identify and clone a new alternatively spliced form of the mGluR1 mRNA. The newly identified mGluR1f mRNA is expressed at moderate levels in rat brain, reaching its maximum in cortex. mGluR1f differs from the mGluR1a mRNA by deletion of a 35-bp fragment of the mGluR1a/α coding sequence and insertion of an 85-bp fragment, found only in mGluR1b/β mRNA.