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1. The metabotropic glutamate receptor 4 (mGluR4) is a Galphai-coupled receptor that modulates glutamatergic neurotransmission. As mGluR4 expression and activation have been implicated in a number of pathological conditions and because the internalization and desensitization properties of this receptor are poorly understood, studies were designed to investigate these aspects of mGluR4 biology. 2. Neither agonist activation by L-(+)-2-amino-4-phosphonobutyric acid (L-AP4) nor L-glutamate caused mGluR4 internalization when cmyc-tagged mGluR4 was expressed in a human embryonic kidney 293 cell line as assessed by cell surface enzyme-linked immunosorbent and immunostaining assays. Instead, a modest increase in mGluR4 surface expression was observed and found to be receptor specific as the competitive antagonist alpha-cyclopropyl-4-phosphonophenylglycine (CPPG) blocked this effect. 3. In contrast, mGluR4 internalized when the protein kinase C (PKC) pathway was activated either by phorbol-12-myristate-13-acetate (PMA) or by the activation of the Galphaq-coupled, neurokinin 3 receptor (NK3R) when co-expressed. This process was PKC-dependent as the specific PKC inhibitor GF 109203X inhibited PMA and NK3R-mediated internalization. 4. PKC activation by PMA caused desensitization of mGluR4 as measured by forskolin-stimulated cAMP inhibition, whereas agonist activation had no effect on desensitization. 5. When mGluR4's coupling was redirected from adenylyl cyclase to phospholipase C by coexpression of a chimeric Galphaqo5 protein, mGluR4 both internalized and desensitized in response to its agonists. 6. These findings demonstrate that mGluR4 internalization and desensitization are agonist-independent unless pathways leading to the activation of PKC are induced.

We have identified 2-methyl-6-(2-phenylethenyl)pyridine (SIB-1893) and 2-methyl-6-phenylethynyl pyridine hydrochloride (MPEP) as positive allosteric modulators for the hmGluR4. SIB-1893 and MPEP enhanced the potency and efficacy of L-2-amino-4-phophonobutyrate (L-AP4) in guanosine 5′-O-(3-[35S]thiotriphosphate ([35S]GTPγS) binding and efficacy in cAMP studies. These effects were fully blocked by the mGluR4 competitive antagonist (RS)-α-cyclopropyl-4-phosphonophenylglycine (CPPG), indicating a dependency on receptor activation. Although SIB-1893 and MPEP had no effects alone in GTPγS binding, effects were observed in the cell-based cAMP assay due to media-derived activation as indicated by CPPG inhibition. Positive modulation of the mGluR4 was a receptor-specific effect since SIB-1893 and MPEP had neither effects on mGluR2-expressing cells nor on the parent BHK cell line. In [3H]L-AP4 binding, a two-fold decrease in KD but not in Bmax was observed with 100 μM SIB-1893, whereas MPEP affected neither parameter. Finally, SIB-1893 and MPEP failed to displace [3H]L-AP4 binding. Taken together, these data identify positive allosteric modulators for the hmGluR4.

1. The metabotropic glutamate receptor 4 (mGluR4) is a Galphai-coupled receptor that modulates glutamatergic neurotransmission. As mGluR4 expression and activation have been implicated in a number of pathological conditions and because the internalization and desensitization properties of this receptor are poorly understood, studies were designed to investigate these aspects of mGluR4 biology. 2. Neither agonist activation by L-(+)-2-amino-4-phosphonobutyric acid (L-AP4) nor L-glutamate caused mGluR4 internalization when cmyc-tagged mGluR4 was expressed in a human embryonic kidney 293 cell line as assessed by cell surface enzyme-linked immunosorbent and immunostaining assays. Instead, a modest increase in mGluR4 surface expression was observed and found to be receptor specific as the competitive antagonist alpha-cyclopropyl-4-phosphonophenylglycine (CPPG) blocked this effect. 3. In contrast, mGluR4 internalized when the protein kinase C (PKC) pathway was activated either by phorbol-12-myristate-13-acetate (PMA) or by the activation of the Galphaq-coupled, neurokinin 3 receptor (NK3R) when co-expressed. This process was PKC-dependent as the specific PKC inhibitor GF 109203X inhibited PMA and NK3R-mediated internalization. 4. PKC activation by PMA caused desensitization of mGluR4 as measured by forskolin-stimulated cAMP inhibition, whereas agonist activation had no effect on desensitization. 5. When mGluR4's coupling was redirected from adenylyl cyclase to phospholipase C by coexpression of a chimeric Galphaqo5 protein, mGluR4 both internalized and desensitized in response to its agonists. 6. These findings demonstrate that mGluR4 internalization and desensitization are agonist-independent unless pathways leading to the activation of PKC are induced.