The Metabotropic Glutamate Receptor mGluR5 Is Endocytosed by a Clathrin-independent Pathway

Abstract

Metabotropic glutamate receptors 5 (mGluR5) are members of the growing group C G protein-coupled receptor family. Widely expressed in mammalian brain, they are involved in modulation of the glutamate transmission. By means of transfection of mGluR5 receptors in COS-7 cells and primary hippocampal neurons in culture followed by immunocytochemistry and quantitative image analysis and by a biochemical assay, we have studied the internalization of mGluR5 splice variants. mGluR5a and -5b were endocytosed in COS-7 cells as well as in axons and dendrites of cultured neurons. Endocytosis occurred even in the absence of receptor activity, because receptors mutated in the glutamate binding site were still internalized as well as receptors in which endogenous activity had been inhibited by an inverse agonist. We have measured a constitutive rate of endocytosis of 11.77/min for mGluR5a. We report for the first time the endocytosis pathway of mGluR5. Internalization of mGluR5 is not mediated by clathrin-coated pits. Indeed, inhibition of this pathway by Eps15 dominant negative mutants did not disturb their endocytosis. However, the large GTPase dynamin 2 is implicated in the endocytosis of mGluR5 in COS-7. mGluR5 is the first shown member of the group C G-protein coupled receptor family internalized by a nonconventional pathway. Metabotropic glutamate receptors 5 (mGluR5) are members of the growing group C G protein-coupled receptor family. Widely expressed in mammalian brain, they are involved in modulation of the glutamate transmission. By means of transfection of mGluR5 receptors in COS-7 cells and primary hippocampal neurons in culture followed by immunocytochemistry and quantitative image analysis and by a biochemical assay, we have studied the internalization of mGluR5 splice variants. mGluR5a and -5b were endocytosed in COS-7 cells as well as in axons and dendrites of cultured neurons. Endocytosis occurred even in the absence of receptor activity, because receptors mutated in the glutamate binding site were still internalized as well as receptors in which endogenous activity had been inhibited by an inverse agonist. We have measured a constitutive rate of endocytosis of 11.77/min for mGluR5a. We report for the first time the endocytosis pathway of mGluR5. Internalization of mGluR5 is not mediated by clathrin-coated pits. Indeed, inhibition of this pathway by Eps15 dominant negative mutants did not disturb their endocytosis. However, the large GTPase dynamin 2 is implicated in the endocytosis of mGluR5 in COS-7. mGluR5 is the first shown member of the group C G-protein coupled receptor family internalized by a nonconventional pathway. metabotropic glutamate receptors γ-aminobutyric acid receptor B G protein-coupled receptor α-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid 2-methyl-6-(phenylethynyl)pyridine green fluorescent protein transferrin rhodamine hemagglutinin phosphate-buffered saline N-hydroxysuccinimide Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. It exerts its effects by binding to ionotropic and metabotropic receptors. Metabotropic glutamate receptors (mGluRs)1 belong to the seven-transmembrane domain G protein-coupled receptor (GPCR) family. Together with calcium-sensing receptors, γ-aminobutyric acid receptor B (GABAB) and pheromone, olfactory and taste receptors, mGluRs form the third family of GPCRs (group C) (for reviews, see Refs. 1Bockaert J. Pin J.P. EMBO J. 1999; 18: 1723-1729Google Scholar and2Hermans E. Challiss R.A. Biochem. J. 2001; 359: 465-484Google Scholar). They have little homology with other GPCRs from groups A and B and are characterized by a large N-terminal extracellular domain that binds glutamate. Eight genes code for mGluRs that generate more than 15 proteins by alternative splicing. On the basis of their sequence similarity, pharmacology, and signal transduction mechanisms, mGluRs have been classified into three groups. Group 1 consists of mGluR1 and mGluR5 that are preferentially positively coupled to phospholipase C via Gq/11 proteins. mGluRs of group 2 and 3 are negatively coupled to adenylate cyclase. Group 1 mGluRs are expressed in neuronal and glial cells within the brain, and receptor activation has been implicated in several forms of synaptic plasticity. Currently, very little is known about the targeting and trafficking mechanisms of mGluRs in cells. During the last few years, attention has focused mainly on endocytosis of a subtype of ionotropic glutamate receptors: the α-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) receptors. Internalization of AMPA receptors in response to various stimuli is an important process for the rapid attenuation of neurotransmission during synaptic plasticity (3Carroll R.C. Beattie E.C. von Zastrow M. Malenka R.C. Nat. Rev. Neurosci. 2001; 2: 315-324Google Scholar). By different approaches, it has been shown that AMPA receptors are endocytosed by a clathrin-coated pit-mediated process (4Carroll R.C. Beattie E.C. Xia H. Luscher C. Altschuler Y. Nicoll R.A. Malenka R.C. von Zastrow M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14112-14117Google Scholar, 5Man Y.H. Lin J.W. Ju W.H. Ahmadian G. Liu L. Becker L.E. Sheng M. Wang Y.T. Neuron. 2000; 25: 649-662Google Scholar, 6Wang Y.T. Linden D.J. Neuron. 2000; 25: 635-647Google Scholar). We are interested in understanding the endocytic properties of mGluR5. Alternative splicing of the mGluR5 gene generates two splice variants: mGluR5a and mGluR5b, which differ only by a 32-amino acid insertion in the intracellular C tail domain of mGluR5b. There is no functional difference between them except that mGluR5b seems to be the predominant form expressed in brain of adult animals (7Romano C. van den Pol A.N. O'Malley K.L. J. Comp. Neurol. 1996; 367: 403-412Google Scholar). Both splice variants as well as mGluR1a interact with the family of Homer proteins (2Hermans E. Challiss R.A. Biochem. J. 2001; 359: 465-484Google Scholar). The first identified member of this family, Homer1a, is a product of an immediate early gene expression, which is induced during intense neuronal activities. All Homers can bind to mGluR5 and mGluR1a by their amino terminus and also to other proteins of the postsynaptic density. Although most GPCRs undergo endocytosis, the conditions and mechanisms of this process can vary from receptor to receptor. Many of them are endocytosed via the clathrin-coated pit pathway but some are not (8Tsao P.I. von Zastrow M. Pharmacol. Ther. 2001; 89: 139-147Google Scholar, 9Dautry-Varsat A. Marsh M. Endocytosis. Oxford University Press Inc., New York2001: 27-57Google Scholar, 10Nichols B.J. Lippincott-Schwartz J. Trends Cell Biol. 2001; 11: 406-412Google Scholar). Some have an agonist-induced endocytosis, and some are continuously endocytosed even in the absence of stimulation. These different behaviors should serve distinct physiological functions. By using transfection of epitope-tagged mGluR5 receptors in COS-7 cells and hippocampal cultured neurons we found that mGluR5a and -5b are endocytosed constitutively by a clathrin-independent pathway. We obtained anti-Myc monoclonal antibody (9E10) from Roche Molecular Biochemicals; anti-Myc polyclonal antibody and anti-mGluR5 from Upstate Biotechnology, Inc. (Lake Placid, NY); anti-HA polyclonal antibody from MBL (Medical and Biological Laboratories Co., Ltd.); anti-MAP2 polyclonal from Peninsula Laboratories Europe Ltd.; anti-α-adaptin monoclonal clone AP6 from Affinity Bioreagents Inc.; and Cy3-, Cy5-, and fluorescein isothiocyanate-conjugated secondary antibodies from Jackson Immunoresearch Laboratories. Dulbecco's modified Eagle's medium, minimum essential medium, F-12/Dulbecco's modified Eagle's medium, Glutamax, and fetal bovine serum came from Invitrogen, and other supplements were from Sigma. 2-Methyl-6-(phenylethynyl)pyridine (MPEP) was a kind gift from Dr. Rainer Kühn (NovartisPharma, Basel, Switzerland). Human holotransferrin was coupled to rhodamine (Rh-Tf) as described in Hémar et al. (11Hémar A. Olivo J.-C. Saffrich R. Williamson E. Dotti C.G. J. Neurosci. 1997; 17: 9026-9034Google Scholar). Sulfo-NHS-SS-biotin was from Pierce, and the streptavidin-agarose beads were from Sigma. The epitope-tagged rat mGluR5a and mGluR5b expression plasmids were constructed using, as a template, the pRKG5a and pRKG5b plasmids that encode mGluR5a and mGluR5b (12Joly C. Gomeza J. Brabet I. Curry K. Bockaert J. Pin J.P. J. Neurosci. 1995; 15: 3970-3981Google Scholar). An MluI restriction site was inserted just after the signal peptide for the N-terminal epitope-tagged receptor (between the Ser22 and Ser23 codons of mGluR5a) using a PCR overlap extension method. Sense and antisense oligonucleotides coding the c-Myc epitope (EQKLISEEDL) or the HA epitope (YPYDVPDYA) with MluI cohesive ends were synthesized and used to introduce the c-Myc or the HA into the N-terminal MluI site. Using this method, we generated the plasmids pRKG5aNmyc (mGluR5-myc in Ref. 13Sergé A. L. Hémar A. J. Neurosci. and and By in cells as described S. Gomeza J. L. Bockaert J. Pin J.P. J. Biol. we that the epitope the G protein the of the receptor mutated in the glutamate binding site were obtained by using the the and were by M. H. Biol. Scholar). the by we used two by A. and A. A. C. A. J. Cell Biol. and dominant negative A. M. A. J. Cell Sci. 1999; and in in in cells the are means of three in in a are means of three in COS-7 cells were in Dulbecco's modified Eagle's with fetal and 2 COS-7 cells were with the different using Molecular to the cells were from rat to the of and K. G. G. K. Scholar). the were by and on a of and in a a 5 of cells. All were in cells in were with plasmids using to the except that the cells were only 1 in with and endocytosis of receptors, antibodies anti-Myc or anti-HA in culture medium, were on cells for was a of were by for in in or antibodies were with an acid with Dulbecco's modified Eagle's medium, cells were for with in in to groups and in bovine serum for the cells were for with anti-MAP2 in three in cells were for with secondary antibodies or fluorescein several with in and in as described by Hémar et al. A. A. M. E. R. A. J. Cell Biol. 1995; Scholar). the anti-Myc was after been on cells for cells were for 1 with secondary antibodies and and in was with a 2 were with a by were with The and of endocytic was by a that an of the The the by and different of analysis Scholar). the by the and are by an neurons or by COS-7 cells. are as the of a between the image and the The are as the of the of was by using a The the COS-7 cells with expression mGluR5a were and in in to in transfection between were in and with 1 of in for two in two were in and the were different in were in well was the other was three for 15 with 1 of and and in All of the were by in 1 of and Molecular on was for of was for an of the of mGluR5 of were with of streptavidin-agarose The beads were three in and in The beads were with The and the beads were by using anti-mGluR5 antibody The was used for analysis of was using We measured the of mGluR5 the receptors the of and intracellular receptors time of and the intracellular receptors receptors to We measured for the of mGluR5 in to The of internalized mGluR5 was with the 1 the receptors or endocytosed receptors which the The endocytosis rate was by to a of the form is using The endocytosis rate is internalization of mGluR5 receptors, receptors were expressed in COS-7 cells or in hippocampal neurons. COS-7 the receptors are the and also in in intracellular of the and endocytic is no antibody the extracellular of we used mGluR5a and mGluR5b the c-Myc or HA epitope the extracellular of receptors on cells with Endocytosis was as cells were for with antibodies were or not with an acid cells were and with fluorescent secondary Both splice and were endocytosed as shown by the of an acid and The are because they antibodies that have been internalized from culture The antibody was internalized by endocytosis, no intracellular was in cells on the A with C with The of the obtained with or acid that are still receptors the with A with The very different from that of the endocytic antibodies were after the conditions and G and The as with the intracellular G with with that the binding of the antibody has not induced a of the receptor the the of the receptors by the anti-Myc antibody not to be for their we the anti-Myc were and A. to the mGluR5 receptors are also endocytosed in we the in primary of rat hippocampal neurons. mGluR5a and are expressed and endocytosed in dendrites and axons Indeed, after anti-Myc as large fluorescent acid in and and and with acid with A and acid The internalized receptors are found in dendrites with the B and and in axons that not C and the conditions we mGluR5a or mGluR5b receptors are endocytosed the of glutamate. that the receptors be endocytosed which means in the absence of binding Although the first have been in the absence of that an is by the cells and the internalization of the receptor. this we studied the endocytosis of mutated receptors that have a for glutamate. We of the of the glutamate binding site of group 1 metabotropic glutamate to the The binding site of mGluR1 has been and the amino with glutamate have been identified Y. Y. M. S. H. K. 2000; Scholar). We mutated into the and the of mGluR5a and The for glutamate in the and mutated receptors is and than that measured with the Both mutated receptors in COS-7 cells were endocytosed A and We have the endocytosis by image We the of acid fluorescent The obtained for mutants are not different from that of the in cells in cells in cells We that the binding of a is not for the endocytosis of mGluR5a. It has been shown that mGluR5 receptors an endogenous constitutive activity that can be inhibited by the inverse A. S. C. M. L. Pin J.P. A. I. R. J. Biol. 2000; Scholar, K. M. I. M. H. R. S. E.C. G. R. 1999; Scholar). also the activity by the to for endocytosis of in the absence of activity, we cells with for and during the anti-Myc in endocytosis 3 that in COS-7 cells and in neurons mGluR5a is endocytosed even in the of The of the are in The of acid fluorescent was not and even that the endocytosis is not inhibited in the of in cells COS-7 with in in with in We that binding activity of the receptor is for endocytosis of mGluR5a. to the of receptors that undergo constitutive endocytosis we of endocytosis on COS-7 cells with mGluR5a with the cells were in for different on streptavidin-agarose beads and with anti-mGluR5 we measured the of and intracellular of receptors. the mGluR5 on The of intracellular receptors with time the the was in this that the receptor is constitutively We the of the and the of internalized receptors see for The time of endocytosis can be well described by a and a time of The rate of endocytosis is that the constitutive endocytosis of mGluR5 is not to endocytosis of the this is than J. 2000; Scholar). internalization can in a The most characterized of endocytosis is but it has been that receptors can the by other in Refs. 9Dautry-Varsat A. Marsh M. Endocytosis. Oxford University Press Inc., New York2001: 27-57Google Scholar and 10Nichols B.J. Lippincott-Schwartz J. Trends Cell Biol. 2001; 11: 406-412Google Scholar). to between the are dominant negative mutants of proteins implicated in endocytosis Eps15 and the of an dominant negative of Eps15 that still the binding site for the clathrin-coated pit and endocytosis A. C. A. J. Cell Biol. Scholar, A. M. A. J. Cell Sci. 1999; Scholar). We used this to see it endocytosis of mGluR5a in COS-7 cells and in hippocampal neurons. We and and for the endocytosis of anti-HA to the of the Eps15 endocytosis was in the cells. endocytosis is and (for COS-7 and C and (for that endocytosis of the receptor is still in cells and that the endocytosis in the of dominant negative of is the as in cells with a Eps15 the binding site A and for for The are for COS-7 2 in cells in cells and for the in cells in cells Eps15 has not been implicated in other than the clathrin-coated pathway L. A. we that mGluR5a is endocytosed a pathway. after endocytosis, the receptor is found in the as transferrin endocytosed by clathrin-coated has been also for the receptor and more for the receptor A. A. M. E. R. A. J. Cell Biol. 1995; Scholar, A. A. A. J. Cell Sci. Scholar, H. J. Biol. is endocytosed in neurons in which clathrin-coated pit internalization is neurons were with with an expression a mutated Eps15 or a Eps15 and for with anti-HA antibodies were and the intracellular antibodies were by secondary A and of B and of endocytosed in the of the of the in neurons as well as in COS-7 cells. the from which the has been that the mGluR5a is not in clathrin-coated the we a of the receptor and of after is a protein that is of the which is with clathrin-coated pit the C. Rev. Cell Biol. 2001; 17: Scholar). very is between the and no was with using antibodies not 1 and 2 are large that have been implicated in and endocytosis A. Marsh M. Endocytosis. Oxford University Press Inc., New York2001: 27-57Google Scholar, 10Nichols B.J. Lippincott-Schwartz J. Trends Cell Biol. 2001; 11: 406-412Google Scholar, L. A. Scholar, J. Cell Biol. Scholar, B.J. J. Cell Biol. Scholar, H. Y. Trends Biochem. Sci. 2000; 25: Scholar). 1 is a protein that is used for the of synaptic dynamin 2 is expressed H. Biol. Scholar). more the used by mGluR5 to be COS-7 cells or cultured hippocampal neurons have been with and dynamin 1 or dynamin 2 or dominant negative mutants coupled to The dominant negative and are in their GTPase activity and transferrin in COS-7 cells H. J. Cell Biol. not COS-7 we still acid intracellular of endocytosed anti-HA antibodies cells are with C and The inhibition in the is not in cells in cells 1 dominant negative is not to mGluR5a internalization in COS-7 it the endocytosis of with endocytosis inhibition in cells in cells after acid was the receptor is the in the of and 2 is for the clathrin-independent endocytosis of mGluR5a. that the dynamin variants not the and be hippocampal dynamin mutants to be well expressed and the mGluR5a is still endocytosed with dynamin 1 or 2 dominant negative in cells in cells in cells in cells by and image that the group 1 metabotropic glutamate receptor mGluR5a is endocytosed in COS-7 cells and in axons and dendrites of hippocampal neurons in culture mGluR5a is endocytosed because mutated receptors that bind glutamate are internalized and because the receptor is still internalized in the of the inverse that the and activity of the receptor We found that the endocytic rate is 11.77/min as measured by a biochemical Using dominant negative mutants of proteins involved in clathrin-coated pit-mediated endocytosis and we that mGluR5a is endocytosed by a clathrin-independent pathway and on the in of mGluR5 a mainly but also of mGluR5 in C. C. O'Malley K. den Pol A. J.W. J. Comp. Neurol. 1995; Scholar, R. R. J. Neurosci. 1996; Scholar). The were with antibodies that not between the two splice variants mGluR5a and that differ only by amino inserted in the intracellular of the mGluR5b (12Joly C. Gomeza J. Brabet I. Curry K. Bockaert J. Pin J.P. J. Neurosci. 1995; 15: 3970-3981Google Scholar, R. H. Biochem. Scholar). is that splice be and the other the first we report that epitope-tagged mGluR5a and mGluR5b are expressed and endocytosed in axons and dendrites of hippocampal neurons in The expression of the mGluR5 receptors in hippocampal neurons in culture with the in hippocampal neurons of which homology with mGluR5 not Neuron. 1999; Scholar, A. J. Neurosci. Scholar). the that mGluR5 splice variants have different et al. Pin J.P. Bockaert J. L. J. Neurosci. 2000; have shown in cultured mGluR5a is expressed only in of the immediate early gene product to the expression of the receptor in dendrites and axons as we in hippocampal neurons. of the forms of and to the expression of mGluR5a. they found in neurons in culture that mGluR5a is expressed in with we have by and that hippocampal neurons in culture Homer proteins A. L. Hémar A. J. Neurosci. not Endocytosis of mGluR5 has been in of cultured neurons M. J. Neurosci. 2000; Scholar). we report that splice variants are endocytosed in cultured neurons from the central nervous in and in endocytosis of receptors is not well to only report the endocytosis of a receptor M. G. 2000; Scholar). et al. I. E. EMBO J. 2001; that the tail of the a sequence to a protein to axons and dendrites of hippocampal but it endocytosis of this only in the to a that endocytosis should have different than that in By two different approaches, we that is endocytosed constitutively in absence of binding and phospholipase C We obtained the for endocytosis of mGluR5b in the of By a biochemical we that the receptor is also constitutively endocytosed a rate with et in the that of the of mGluR5 is they have not identified the of intracellular or endocytic M. Y. J. Neurosci. 2001; Scholar). Although we a constitutive endocytosis, it not that endocytosis is not by or receptor A of mGluR5 endocytosis by has been described in neurons M. J. Neurosci. 2000; Scholar). It has been by several groups that the very of is endocytosed in cells. Both a constitutive and a endocytosis have been R.A. 1997; Scholar, Biochem. J. 1999; Scholar, G. E. J. 2001; Scholar, M. Pharmacol. 2001; Scholar, J. M. M. Scholar). We the of intracellular is after from an endocytosis, a of the of in the intracellular pathway followed by mGluR5. We the biochemical in to of endocytosis of that we by image the we the of but not the and of The constitutive endocytosis of mGluR5 an intracellular that the to the of receptors. Using the dominant negative of a protein involved in clathrin-coated pit-mediated endocytosis, we that mGluR5a is endocytosed by a pit pathway. The have been obtained for mGluR5b not for the first time that a member of group C is constitutively endocytosed by a nonconventional pathway. mGluR1a endocytosis in cells seems to clathrin-coated G. E. J. 2001; Scholar, J. M. M. the constitutive endocytosis pathway is well M. Pharmacol. 2001; Scholar). have the of because the endocytosis was inhibited by and because the receptor is found in the as not only endocytosis. it and receptors endocytosis that are not mediated by clathrin-coated J. Cell Sci. 2001; Scholar, H. 2001; Scholar). mGluR5 endocytosis is also inhibited by in COS-7 cells not and endocytosed receptors with endocytosed transferrin et al. M. Pharmacol. 2001; that 1 dominant negative not mGluR1a endocytosis as we found for mGluR5. using Eps15 mutants on mGluR1a constitutive internalization be more for the understanding of the constitutive internalization pathway followed by mGluR5a well as not endocytosis is to dynamin 1 in COS-7 cells and in neurons. However, in COS-7 the nonconventional pathway followed by mGluR5 dynamin the endogenous in cells. dynamin 1 dominant negative is not to with dynamin 2 in this it with dynamin 2 implicated in clathrin-coated pit endocytosis. Indeed, transferrin endocytosis is inhibited by dynamin 1 and dynamin 2 dominant negative mutants in COS-7 cells not H. Biol. Scholar). that have distinct in and endocytosis. can be with the of et Y. K. S. J. Cell Biol. a different of in endocytosis in the cells. in a with the of we have shown that the dynamin H. Y. Trends Biochem. Sci. 2000; 25: can mGluR5 endocytosis in COS-7 cells not we obtained a different in that dynamin 2 dominant negative mutants not mGluR5 endocytosis, 2 mutants are to the to the site of mGluR5 endocytosis the is not to neurons. be because is only using in neurons. The that dynamin 2 mutants the ionotropic glutamate receptor AMPA internalization (4Carroll R.C. Beattie E.C. Xia H. Luscher C. Altschuler Y. Nicoll R.A. Malenka R.C. von Zastrow M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14112-14117Google Scholar). for an in hippocampal neurons is that an endocytosis pathway used by mGluR5 that not on 1 and the of in the different neuronal they are expressed is not except for dynamin 1 in synaptic Nat. Rev. Neurosci. 2000; Scholar, C. J. J. Biol. 2001; Scholar). The of in pit-mediated endocytosis has been described in cells J. Cell Biol. Scholar, B.J. J. Cell Biol. C. A. C.G. A. A. 2001; Scholar, Y. van J. Biol. 2000; Scholar). Endocytosis of some GPCRs is to dynamin 1 dominant negative mutants in cells the receptor C. van J. Biol. receptor von Zastrow M. J. Cell Biol. 1999; and receptor J. L. J. Biol. 1996; that they are not endocytosed by a clathrin-coated pit-mediated endocytosis. It be to the pathway used by receptors dynamin 2 as we for mGluR5. An of dynamin 1 dominant negative mutants has been in some pit in the of receptor endocytosis using the C. A. C.G. A. A. 2001; and in the of receptor using dynamin 1 that the binding domain or the GTPase activity Y. van J. Biol. 2000; Scholar). It is to that in that last the dynamin 1 was not to endocytosis. that not interact with the in and endocytosis. be this clathrin-independent The studied pathway is endocytosis, but few receptors have been found in have not been in and in neurons M. M. J. 1999; Scholar, K. Nat. Rev. Biol. 2000; Scholar, Cell 2001; Scholar). A endocytosis be are in and in which is of proteins and some K. Nat. Rev. Biol. 2000; Scholar). receptors have been in and in of the A first to the internalization pathway of mGluR5 be to mGluR5 is in There are two the the G protein coupled to is in S. J. Biol. 2000; Scholar, Biol. 2001; and endocytosed clathrin-coated is not found in A. R.A. J. 2001; Scholar). of this endocytosis is of for understanding the for endocytosis of receptors. We Rainer and for the of and We are to and for the