Complex neuronal functions rely upon the precise sorting, targeting, and restriction of receptors to specific synaptic microdomains. Little is known, however, of the molecular signals responsible for mediating these selective distributions. Here we report that metabotropic glutamate receptor subtype 7a (mGluR7a) is polarized at the basolateral surface when expressed in Madin-Darby canine kidney (MDCK) epithelial cells but is not polarized when expressed in cultured hippocampal neurons. Truncation of the mGluR7 cytoplasmic tail produces a protein that is restricted to a perinuclear intracellular compartment in both neurons and MDCK cells, where this protein colocalizes with a trans-Golgi network antigen. The mGluR7 cytoplasmic domain appended to the transmembrane portion of the vesicular stomatitis virus G protein and the ectodomain of human placental alkaline phosphatase is distributed over the entire cell surface in cultured neurons. When expressed in MDCK cells, this construct remains in an intracellular compartment distinct from endosomes or lysosomes. Thus, the cytoplasmic tail domain of mGluR7 is necessary but not sufficient for polarized targeting in MDCK monolayers, whereas in neurons the cytoplasmic tail is sufficient for cell surface expression but not polarization. Additional mechanisms are likely required to mediate mGluR7 neuronal polarization and synaptic clustering. Complex neuronal functions rely upon the precise sorting, targeting, and restriction of receptors to specific synaptic microdomains. Little is known, however, of the molecular signals responsible for mediating these selective distributions. Here we report that metabotropic glutamate receptor subtype 7a (mGluR7a) is polarized at the basolateral surface when expressed in Madin-Darby canine kidney (MDCK) epithelial cells but is not polarized when expressed in cultured hippocampal neurons. Truncation of the mGluR7 cytoplasmic tail produces a protein that is restricted to a perinuclear intracellular compartment in both neurons and MDCK cells, where this protein colocalizes with a trans-Golgi network antigen. The mGluR7 cytoplasmic domain appended to the transmembrane portion of the vesicular stomatitis virus G protein and the ectodomain of human placental alkaline phosphatase is distributed over the entire cell surface in cultured neurons. When expressed in MDCK cells, this construct remains in an intracellular compartment distinct from endosomes or lysosomes. Thus, the cytoplasmic tail domain of mGluR7 is necessary but not sufficient for polarized targeting in MDCK monolayers, whereas in neurons the cytoplasmic tail is sufficient for cell surface expression but not polarization. Additional mechanisms are likely required to mediate mGluR7 neuronal polarization and synaptic clustering. rough endoplasmic reticulum trans-Golgi network Madin-Darby canine kidney epithelial cells polymerase chain reaction green fluorescent protein wild type Dulbecco's modified Eagle's medium metabotropic glutamate receptor subtype 7a placental alkaline phosphatase vesicular stomatitis virus G protein Specialized domains at the neuronal cell surface orchestrate complex synaptic functions. Accurate sorting, trafficking, and restriction of proteins to these microdomains ensures the precise distribution of synaptic proteins for proper neuronal function. Proteins destined for secretion or incorporation into the plasma membrane transit to the cell surface through a series of subcellular compartments. Synthesis occurs in the soma at the rough endoplasmic reticulum (RER),1 and postsynthetic processing continues in the Golgi complex, with sorting of proteins into specific transport vesicles occurring in the TGN (1Trimmer J.S. Neuron. 1999; 22: 411-417Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar,2Griffiths G. Simons K. Science. 1986; 234: 438-443Crossref PubMed Scopus (761) Google Scholar). Information contained within targeted proteins function as sorting signals to specify their ultimate destination within the cell (3Caplan M.J Am. J. Physiol. 1997; 272: F425-429Crossref PubMed Google Scholar, 4Muth T.R. Ahn J. Caplan M.J. J. Biol. Chem. 1998; 273: 25616-25627Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Although progress is being made in the categorization of sorting signals, their diversity and complexity, as well as the underlying mechanisms through which cells carry out polarized targeting, remain unresolved (1Trimmer J.S. Neuron. 1999; 22: 411-417Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Neurotransmitter receptors are among the complex array of proteins at synaptic microdomains that participate in the generation and propagation of neuronal activity. Glutamate serves as the principle excitatory amino acid neurotransmitter at central nervous system synapses. Activity at glutaminergic synapses is mediated by both ionotropic and metabotropic glutamate receptors (5Hollman M. Heinemann S. Annu. Rev. Neurosci. 1994; 17: 31-108Crossref PubMed Scopus (3660) Google Scholar). Metabotropic glutamate receptors couple with heterotrimeric G-proteins to regulate cell excitability and synaptic transmission (6Pin J-P. Duvoisin R. Neuropharmacol. 1995; 34: 1-26Crossref PubMed Scopus (1234) Google Scholar, 7Conn P.J. Pin J-P. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 205-237Crossref PubMed Scopus (2726) Google Scholar) and have been implicated in synaptic plasticity (8Riedel G. Trends Neurosci. 1996; 19: 219-224Abstract Full Text PDF PubMed Scopus (140) Google Scholar). Currently eight distinct mGluR subtypes have been recognized (6Pin J-P. Duvoisin R. Neuropharmacol. 1995; 34: 1-26Crossref PubMed Scopus (1234) Google Scholar). These have been observed to exhibit subtype- and region-specific differences in distribution along the neuronal cell surface in vivo. The mGluR7 subtype, in particular, highlights the complexity of protein targeting. mGluR7 receptors have been reported to be targeted to postsynaptic sites in the olfactory bulb (9Kinzie J.M. Shinohara M.M. van den Pol A.N. Westbrook G.L. Segerson T.P. J. Comp. Neurol. 1997; 385: 372-384Crossref PubMed Scopus (37) Google Scholar) and locus coeruleus (10Kinoshita A. Shigemoto R. Ohishi H. van der Putten H. Mizuno N. J. Comp. Neurol. 1998; 393: 332-352Crossref PubMed Scopus (241) Google Scholar, 11Bradley S.R. Rees H.D. Yi H. Levey A.I. Conn P.J. J. Neurochem. 1998; 71: 636-645Crossref PubMed Scopus (71) Google Scholar), to axons in the hippocampus (12Bradley S.R. Levey A.I. Hersch S.M. Conn P.J. J. Neurosci. 1996; 16: 2044-2056Crossref PubMed Google Scholar, 13Shigemoto R. Kinoshita A. Wada E. Nomura S. Ohishi H. Takada M. Flor P.J. Neki A. Abe T. Nakanishi S. Mizuno N. J Neurosci. 1997; 17: 7503-7522Crossref PubMed Google Scholar), to both pre- and postsynaptic sites in the retina (14Brandstatter J.H. Koulen P. Kuhn R. van der Putten H. Wassle H. J. Neurosci. 1996; 16: 4749-4756Crossref PubMed Google Scholar), and to both axons and dendrites of cultured hippocampal neurons (15Stowell J.N. Craig A.M. Neuron. 1999; 22: 525-536Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). In the hippocampal formation additional complexity is evident, because the distribution of mGluR7 is apparently dependent upon the nature of the postsynaptic target neuron. Within individual axons, mGluR7 is found to be present only in distinct subpopulations of presynaptic terminals (16Shigemoto R. Kulik A. Roberts J.D.B. Ohishi H. Nusser Z. Kaneko T. Somogyi P. Nature. 1996; 381: 523-524Crossref PubMed Scopus (337) Google Scholar). The subtype- and region-specific placement of mGluR7 at distinct membrane domains requires the existence of mechanisms whereby sorting information is interpreted, so that receptors can be distributed precisely. mGluR7 is a member of the larger seven transmembrane or G-protein-coupled receptor family of receptors. Little is known about the cellular mechanisms that govern the intracellular trafficking of the individual members of this critically important protein family. Because of the technical difficulties inherent in studying sorting and trafficking in neurons, cultured cell lines have been utilized as a means both to study sorting in a polarized cell system and to allow biochemical investigations of the underlying cellular mechanisms. Among these culture models, epithelial cells have been especially useful in studies of neuronal membrane protein targeting (17Rodriguez-Boulan E. Powell S. Annu. Rev. Cell Biol. 1992; 8: 395-427Crossref PubMed Scopus (354) Google Scholar). This utility may be in part based upon the developmental origin of neurons from neuroepithelial stem cells (18Colman D.R. Neuron. 1999; 23: 649-651Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). In some but not all instances, Madin-Darby canine kidney epithelial cells (MDCK) and neurons seem to employ similar signals and mechanisms to generate polarized protein distributions (as discussed in Refs. 4Muth T.R. Ahn J. Caplan M.J. J. Biol. Chem. 1998; 273: 25616-25627Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 15Stowell J.N. Craig A.M. Neuron. 1999; 22: 525-536Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, and 19Lim S.T. Antonucci D.E. Scannevin R., H. Trimmer J.S. Neuron. 2000; 25: 385-397Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Many neuronal proteins found in axons can sort to the apical surface of epithelia, whereas most dendritic proteins can sort to the basolateral surface. We report here an analysis of the subcellular targeting of the mGluR7 receptor. By coordinated investigation in both epithelial cell lines and in cultured hippocampal neurons, we are able to examine whether the requirements for the polarized sorting of the protein are the same in both cell types. We report that mGluR7 achieves a polarized steady-state distribution at the basolateral membrane when expressed in epithelial monolayers. The cytoplasmic tail domain of mGluR7 is necessary but not sufficient for this polarized targeting in MDCK cells. In neurons however, exogenously expressed mGluR7 does not achieve a polarized distribution. The cytoplasmic tail is not sufficient for polarization, but without it the protein does not achieve a cell surface distribution. Cloned mGluR7a cDNA in the pBluescript plasmid was a kind gift from S. Nakanishi (20Masu M. Tanabe Y. Tsuchida K. Shigemoto R. Nakanishi S. Nature. 1991; 349: 760-765Crossref PubMed Scopus (990) Google Scholar). AnXbaI site was introduced at the Bluescript polylinkerNotI site by insertion of an oligonucleotide linker. ThisXbaI site was used along with the pBluescript polylinkerClaI restriction site and the AatII restriction site within mGluR7a for subcloning and fusion protein construction. Fusion PCR was used to construct the GFP·mGluR7 fusion protein. PCR was used to generate three products from which the final fusion products were made by use of the same primers. The first PCR product was generated from the mGluR7 wt cDNA using a 5′-primer complimentary to and including the pBluescript polylinker ClaI restriction site and a 3′-primer that included both the last five codons before the intended site of GFP incorporation (10 amino acids following the signal sequence) and the first seven codons of the GFP sequence. The GFP sequence was generated in a similar manner from pEGFP-C1 (CLONTECH, Palo Alto Ca.) using primers that included both the mGluR7 sequence before the site of intended insertion and the sequence on either end of the GFP encoding sequence. The downstream mGluR7 product was generated with a 5′-primer including both the final five codons of GFP and the initial six codons following the insertion site and a 3′-primer past the AatII restriction site in The first PCR products were and to primers were and the fusion product was This initial fusion product was used in a similar manner with the PCR product from the initial to the fusion PCR The was at the ClaI and AatII restriction sites for into the cDNA plasmid of The construct was generated by PCR with a 5′-primer at the of the GFP and mGluR7 fusion and a 3′-primer selective to a sequence following the last transmembrane The was to a amino acids following the final transmembrane domain and an restriction The was at the and AatII restriction sites for into the cDNA plasmid of The fusion construct was generated using a plasmid M.J. Caplan M.J. Am. J. Physiol. 1995; PubMed Google Scholar). PCR the cytoplasmic tail of mGluR7 was generated using a 5′-primer following the transmembrane domain to and including a restriction and a 3′-primer to an restriction site following the The was at the restriction sites for into the cDNA plasmid of were through the The were into the expression by M. of the ClaI sites to cells were in Dulbecco's modified Eagle's medium with and MDCK cells were in with and were in a with and through to cells were using J. T.R. G. Caplan M.J. J. Biol. Chem. 1996; Full Text Full Text PDF Scopus Google Scholar). were on the before were in and to of and was for at The was and the cells were in of and for at The cells were in and in for to MDCK cells were as J. T.R. G. Caplan M.J. J. Biol. Chem. 1996; Full Text Full Text PDF Scopus Google Scholar) and by the with fusion that been into the This to the in were for expression by or phosphatase hippocampal were as S.T. Antonucci D.E. Scannevin R., H. Trimmer J.S. Neuron. 2000; 25: 385-397Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). neurons were at a when synapses have been and synaptic receptors have polarized and to the synapses. hippocampal neurons were at in by the In of was into of medium and at for of was into of medium in a The and the were and for at were to a that contained of the neuronal medium from the hippocampal The were into the medium the cells and at with for The were into the and for was as T.R. Ahn J. Caplan M.J. J. Biol. Chem. 1998; 273: 25616-25627Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) on cell cells were to on cell culture for on and in for at were in a and for of was by the cells in at for were either by GFP wt by J. S.R. Levey A.I. Hersch S.M. Conn P.J. J. Neurosci. 1996; 16: 2044-2056Crossref PubMed Google Scholar), or or were to either or in for at and the cells were to three in the with the cells were in three for and in for before being on with were using a are the product of were generated with a and were so that all were in the of neurons was as S.T. Antonucci D.E. Scannevin R., H. Trimmer J.S. Neuron. 2000; 25: 385-397Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). were with on a was as Biol. 1998; PubMed Scopus Google Scholar), cells were for with in both and were on for and the medium was with before the of either apical or basolateral proteins was out at as J. T.R. G. Caplan M.J. J. Biol. Chem. 1996; Full Text Full Text PDF Scopus Google Scholar). were with for were from the with a and the were in in The proteins apical or basolateral cell surface were from cell by with of proteins were from the in by and as J. T.R. G. Caplan M.J. J. Biol. Chem. 1996; Full Text Full Text PDF Scopus Google Scholar). The was with or by and with mGluR7 is a domain receptor with a domain and a acid cytoplasmic The construct wt mGluR7 was by use of a specific S.R. Rees H.D. Yi H. Levey A.I. Conn P.J. J. Neurochem. 1998; 71: 636-645Crossref PubMed Scopus (71) Google Scholar) the cytoplasmic tail of investigations we to the receptor by GFP·mGluR7 fusion The fusion protein construct GFP in at a amino acids to the signal sequence investigations of the of the cytoplasmic tail in receptor the cytoplasmic tail was from the fusion protein at a amino acids to the end of the final transmembrane domain the construct tail whether the cytoplasmic tail of mGluR7 may function to a fusion protein was generated with the mGluR7 cytoplasmic tail with a construct the transmembrane domain and placental alkaline phosphatase as the were expressed in cells. wt and a cell surface distribution in cells. MDCK cells expressed mGluR7 at the plasma MDCK cell lines mGluR7 were to and by and using the for wt and either GFP or for a polarized steady-state distribution at the basolateral surface of MDCK epithelial cells and the polarized distributions of the mGluR7 we the cell surface to MDCK apical and basolateral cell surface the by distribution at the basolateral surface distribution of and mGluR7 expressed in polarized MDCK cells by MDCK cells were with mGluR7 construct in the expression and of MDCK cells were an intracellular distribution of mGluR7 wt as by with the intracellular distribution of mGluR7 is The GFP·mGluR7 receptor construct a basolateral cell surface distribution in MDCK cells as by GFP intracellular mGluR7 is observed In however, of GFP·mGluR7 with the cytoplasmic a intracellular distribution as observed in of GFP·mGluR7 with both and additional intracellular as with The intracellular mGluR7 is distribution of mGluR7 and receptor in polarized MDCK cells as by MDCK cells were with and in the expression of MDCK cells are In a and of the construct to be contained in a intracellular compartment as by with In and of the construct to be contained in a intracellular which a perinuclear distribution similar to that in mGluR7 is expressed at the basolateral cell surface whereas the and are not expressed at the surface of MDCK cells. cell surface analysis of mGluR7 receptor in MDCK cell lines was out using either or to the is as a protein of at the basolateral cell surface a signal is at the apical cell surface the of the construct at the surface of MDCK cells. The construct was not at either the apical or the basolateral cell with intracellular distribution cell surface analysis not the construct at either the apical or basolateral cell that the MDCK cells the analysis was on cell the of a protein of to the examine neurons sort and target we whether the construct is targeted and polarized at in cultured neurons. of the GFP fusion construct of only exogenously expressed the of the signal for was intracellular or was found at the cell surface both axons and dendrites and with clustering. expressed mGluR7 not achieve a polarized distribution in cultured hippocampal neurons and was found at apparently at the cell of both axons and dendrites a of wt and fusion protein distributions that the of these proteins participate in a In MDCK cells the protein distribution to be intracellular when is using an the final amino acids of the mGluR7 when using the same the distribution of to be intracellular In of the GFP signal in the same with cell surface a cell surface distribution the basolateral These that in MDCK cells, the portion of cell surface mGluR7 is with a protein or proteins that with of the to to this the of mGluR7 intracellular not participate in these their in the of the cell with is with this intracellular in to the at the cell surface. that incorporation of mGluR7 into the basolateral plasma membrane of MDCK cells is most likely with the formation of Because the mGluR7 tail to participate in at the MDCK cell basolateral we whether the tail information to protein this we the distributions of the and the expression in cells, to perinuclear intracellular and distribution are of the Golgi complex When these are expressed in MDCK cells, the construct cell distribution and was found in a perinuclear intracellular compartment and the intracellular compartment in which the construct we with an specific to a expressed in the TGN by A. G. A. P. R. E. Cell Biol. 2000; PubMed Scopus Google Scholar). In both cells and in MDCK cells and we found that colocalizes with the TGN In the Golgi of this construct does not seem to be able to to the cell surface and to the TGN as been for and In the of and to the TGN requires and is by that S. J. 1994; PubMed Scopus Google Scholar). with not distribution in MDCK cells not The perinuclear distribution not and not to a distribution Thus, the construct to be a of the TGN that does not from the cell surface When expressed in cells, the construct a cell surface distribution the construct not a cell surface distribution in MDCK cells. it was in a intracellular distribution a and This compartment is not by to the membrane protein that it does not by that this compartment is in cell surface of MDCK that were present at the cell surface and in neurons was to neurons the and at with and of only exogenously expressed In to in MDCK cells, the signal for that was expressed at the neuronal cell surface both axons and was not distributed in a polarized manner and and when exogenously expressed in neurons, was observed in a perinuclear intracellular distribution with a with at the cell surface function upon the polarized distribution of synaptic The mGluR7 subtype is a protein distributed with in neurons, being found only in selective at cell surface synaptic In mGluR7 can be found distributed in either pre- or postsynaptic In particular, mGluR7 to selective subpopulations of terminals within individual neurons of the expression studies have the distribution of mGluR7 expressed in cultured hippocampal neurons (15Stowell J.N. Craig A.M. Neuron. 1999; 22: 525-536Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). that mGluR7 was present at the cell surface of both axons and that the exogenously expressed protein not achieve a polarized distribution. The of this study that the of mGluR7 an targeting because it to The molecular nature of the mGluR7 sorting signal remains We to examine the signals and mechanisms in mGluR7 this we expressed mGluR7 and a of receptor in neurons and epithelial cells. These included a GFP fusion construct of mGluR7 without cytoplasmic and a construct where the cytoplasmic tail of mGluR7 was appended to a transmembrane protein. with the of and Craig (15Stowell J.N. Craig A.M. Neuron. 1999; 22: 525-536Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), we that in cultured hippocampal neurons, mGluR7 is expressed in a distribution at the of both and in MDCK epithelia, exogenously expressed mGluR7 is polarized at the basolateral cell surface. that the cytoplasmic tail of mGluR7 was necessary for surface in both and in neurons but was not sufficient to mediate a polarized distribution in either been that epithelial cells and neurons can use similar signals and mechanisms to sort membrane proteins to polarized distributions at the cell because some neuronal proteins found in axons can sort to the apical surface of epithelia, whereas dendritic proteins can sort to the basolateral surface J. T.R. G. Caplan M.J. J. Biol. Chem. 1996; Full Text Full Text PDF Scopus Google Scholar, Simons K. Full Text PDF PubMed Scopus Google Scholar, G. G. Caplan M.J. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). In the of mGluR7 this is not the mechanisms in neurons that the mGluR7 sorting information from the mechanisms that this information in MDCK cells. have been with a of including and A. M. S. R. J. Cell 1999; PubMed Google Scholar, Ahn J. Caplan M.J. Levey A.I. G. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). We have that the cytoplasmic tail of mGluR7 is necessary for surface because the construct in a perinuclear intracellular and not the cell surface in either MDCK cells or neurons. This compartment colocalizes with a trans-Golgi network in both and MDCK cells. that the cytoplasmic construct to the The of this Golgi is not We can however, that the protein does not to and cell surface This is observed for proteins in the including and TGN protein from the TGN to the cell surface been to as either targeting signals A. N. K. J. 1998; 17: PubMed Scopus Google Scholar) or for of the of protein E. A. Trends Cell Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In the of sites remain Thus, the of may be to an to participate in important for surface In the cytoplasmic tail of mGluR7 is not sufficient to mediate polarized sorting in epithelia, where the construct within the cell in a cytoplasmic distinct from or compartments. In neurons however, this construct was present at the surface of both axons and as was found for exogenously expressed but not achieve a polarized distribution. Thus, the of mGluR7 to cell surface expression is dependent upon the cell type in which are The mGluR7 cytoplasmic tail is sufficient for cell surface in neurons, but not in MDCK cells. this portion of the protein is not by sufficient to specify polarized targeting in either cell is to that the cytoplasmic of a of with proteins A. M. Nature. 1999; PubMed Scopus Google Scholar). Cell surface may be to polarized sorting Physiol. Rev. 1999; PubMed Scopus Google Scholar). with have been implicated not only in cell surface that proteins at specific A. R. S.M. E. A. G. J. 1999; PubMed Scopus Google Scholar), but at of protein targeting at the TGN R. Neuron. 1999; 23: Full Text Full Text PDF PubMed Scopus Google Scholar). This is with that the cytoplasmic tail is in MDCK cells, where mGluR7 is distributed in a polarized but not in cells, which polarized membrane of proteins with the tail of mGluR7 to the construct at the basolateral surface with an to the cytoplasmic In the three final of mGluR7 may a Z. J. S.M. A. J.M. Science. 1997; PubMed Scopus Google Scholar). the of or for mGluR7 not not a or with the MDCK as been with proper of some membrane proteins in MDCK P.J. J. Cell Biol. 1986; Scopus Google Scholar, H. Trimmer J.S. J. Cell Biol. 1996; PubMed Scopus Google Scholar). In the cytoplasmic tail of mGluR7 is necessary for the surface of the receptor but not sufficient to mediate polarized distribution in or in neurons. domains of the mGluR7 be in to sorting The nature of these the signals and the that remain to be We J. P. A. and S. Nakanishi for the and
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