Is the heterologous expression of metabotropic glutamate receptors (mGluRs) an appropriate method to study the mGluR function? Experience with human embryonic kidney 293 cells transfected with mGluR1

The cloning of metabotropic glutamate receptors (mgluRs) has initiated a new approach to the study of their function: the introduction of mGluR cDNA into cells that do not normally express mGluRs, thus allowing the heterologous receptor expression. We have transfected human embryonic kidney (HEK) 293 cells with the full length mGluRla cDNA and with its truncated variant which encodes the receptor termed mGluRlT (a receptor lacking the long intracellular domain and similar to the splice variant mGluR1c). Transient transfection of HEK-293 cells with mGluR1a, but not the mGluR1T cDNA, resulted in a significant increase in inositol phosphate (IP) formation in absence of any mGluR agonists. This effect was completely dependent on the presence of extracellular calcium, and unlike the agonist-stimulated IP formation it was insensitive to pertussis toxin. The prolonged activation of IP formation might affect the cell physiology. In an attempt to obtain stably transfected cells, we transfected about 1.5 × 10⁶ HEK-293 cells with the plasmide conveying the full-length mGluR1a cDNA and the neomicin-resistance gene. Only 12 clones survived the antibiotic selection, and only one of these 12 clones continued to divide. The size of mRNA from the clone was smaller than the full-length mGluR1a mRNA. The shortened mRNA, revealed in the clone, apparently encoded a functional mGluR that was sensitive to glutamate, but unlike the mGluR1a, it did not respond to 1S,3R-ACPD (1S,3R-aminocyclopentane-1,3-dicarboxylic acid). A prudent use of the heterologous cell transfection technique is necessary in studying the xfunction and the pharmacology of mGluRs.     

Agonist-induced internalization of the metabotropic glutamate receptor 1a is arrestin- and dynamin-dependent

At present, little is known regarding the mechanism of metabotropic glutamate receptor (mGluR) trafficking. To facilitate this characterization we inserted a haemagglutinin (HA) epitope tag in the extracellular N-terminal domain of the rat mGluR1a. In human embryonic kidney cells (HEK293), transiently transfected with HA-mGluR1a, the epitope-tagged receptor was primarily localized to the cell surface prior to agonist stimulation. Following stimulation with glutamate (10 microM; 30 min) the HA-mGluR1a underwent internalization to endosomes. Further quantification of receptor internalization was provided by ELISA experiments which showed rapid agonist-induced internalization of the HA-mGluR1a. To determine whether agonist-induced mGluR1a internalization is an arrestin- and dynamin-dependent process, cells were cotransfected with HA-mGluR1a and either of these dynamin-K44A or arrestin-2 (319-418). Expression of either dominant negative mutant constructs with receptor strongly inhibited glutamate-induced (10 microM; 30 min) HA-mGluR1a internalization. In addition, wild-type arrestin-2-green fluorescent protein (arrestin-2-GFP) or arrestin-3-GFP underwent agonist-induced translocation from cytosol to membrane in HEK293 cells coexpressing HA-mGluR1a. Taken together our observations demonstrate that agonist-induced internalization of mGluR1a is an arrestin- and dynamin-dependent process.     

An N-terminal sequence specific for a novel Homer1 isoform controls trafficking of group I metabotropic glutamate receptor in mammalian cells

Homer proteins bind to a proline-rich region of the group I metabotropic glutamate receptors (mGluRs) and control their expression and localization at the excitatory postsynaptic density. We isolated a novel isoform of Homer1, Homer1d, from a mouse heart cDNA library. Its N-terminal end of 18 amino acids was unique among Homer1 variants (Homer1a-d), while the remainder of Homer1d was identical to that of Homer1b. To clarify the function of its N-terminus, we expressed Homer1b and 1d in the presence and absence of mGluR5b in HEK293T cells. When expressed alone, both Homer proteins were distributed diffusely in the cytoplasm and mGluR5b was on the plasma membrane (PM). When co-expressed, Homer1d and mGluR5b were co-localized on the PM, while Homer1b and mGluR5b were retained in the endoplasmic reticulum (ER). Both Homer proteins bound to mGluR5b in vitro. Therefore, the N-terminal portion of Homer1d may facilitate trafficking of Homer1-mGluR5 complex from the ER to the PM.     

Molecular characterisation of two structurally distinct groups of human homers, generated by extensive alternative splicing

Homer proteins bind specifically to the C termini of the metabotropic glutamate receptor mGluR1alpha/a and mGluR5, play a role in their targeting and modulate their synaptic properties. We have discovered that extensive alternative splicing generates a family of 17 Homer proteins. These fall into two distinct groups of 12 "long" Homers, which all have a coiled-coil domain at their C termini, and five "short" Homers, which lack such a domain. All Homers contain the N-terminal sequence responsible for their binding to mGluR1alpha/a receptors and can be co-localised with the recombinantly expressed mGluR1alpha/a protein in HEK-293 cells. The existence of the long and the short variants of each of the Homer-1, Homer-2 and Homer-3 proteins reflects the fundamental principles of Homer functions.     

Phosphorylation-independent desensitization of metabotropic glutamate receptor 5 by G protein-coupled receptor kinase 2 in HEK 293 cells

The metabotropic glutamate receptor 5 (mGluR5) is one of the important excitatory neurotransmitter receptors in the central nervous system, and its desensitization by G protein-coupled receptor kinases (GRKs) plays an important role in neuron protection against receptor overstimulation. It is reported that GRK2 could down-regulate the mGluR5 signaling in both HEK 293 cells and neurons. However, whether GRK2-mediated mGluR5 desensitization is phosphorylation dependent remains controversial. Here, we demonstrated that the signal intensity and kinetics of mGluR5 desensitization was inhibited or changed by GRK2 in HEK 293 cells. By using the catalytically inactive GRK2 mutant K220R, and the receptor mutants that lack potential phosphorylation sites in the C-terminal tail, we demonstrated that the GRK2-mediated mGluR5 desensitization was phosphorylation-independent. Furthermore, overexpression of an N-terminal regulator of G protein signaling (RGS) homology (RH) domain of GRK2 was sufficient to attenuate the mGluR5 signaling, whereas the expression of GRK2 D110A mutant devoid in Gαq binding was unable to inhibit mGluR5 signaling. In summary, this study provides evidence that GRK2 mediates phosphorylationindependent mGluR5 desensitization via the interaction between the RGS domain and Gαq in HEK 293 cells.     

EDEM3, a soluble EDEM homolog, enhances glycoprotein endoplasmic reticulum-associated degradation and mannose trimming

Quality control in the endoplasmic reticulum ensures that only properly folded proteins are retained in the cell through mechanisms that recognize and discard misfolded or unassembled proteins in a process called endoplasmic reticulum-associated degradation (ERAD). We previously cloned EDEM (ER degradation-enhancing alpha-mannosidase-like protein) and showed that it accelerates ERAD of misfolded glycoproteins. We now cloned mouse EDEM3, a soluble homolog of EDEM. EDEM3 consists of 931 amino acids and has all the signature motifs of Class I alpha-mannosidases (glycosyl hydrolase family 47) in its N-terminal domain and a protease-associated motif in its C-terminal region. EDEM3 accelerates glycoprotein ERAD in transfected HEK293 cells, as shown by increased degradation of misfolded alpha1-antitrypsin variant (null (Hong Kong)) and of TCRalpha. Overexpression of EDEM3 also greatly stimulates mannose trimming not only from misfolded alpha1-AT null (Hong Kong) but also from total glycoproteins, in contrast to EDEM, which has no apparent alpha1,2-mannosidase activity. Furthermore, overexpression of the E147Q EDEM3 mutant, which has the mutation in one of the conserved acidic residues essential for enzyme activity of alpha1,2-mannosidases, abolishes the stimulation of mannose trimming and greatly decreases the stimulation of ERAD by EDEM3. These results show that EDEM3 has alpha1,2-mannosidase activity in vivo, suggesting that the mechanism whereby EDEM3 accelerates glycoprotein ERAD is different from that of EDEM.     

Conserved high affinity ligand binding and membrane association in the native and refolded extracellular domain of the human glycine receptor alpha1-subunit

The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated chloride channel composed of ligand binding alpha- and gephyrin anchoring beta-subunits. To identify the secondary and quaternary structures of extramembraneous receptor domains, the N-terminal extracellular domain (alpha1-(1-219)) and the large intracellular TM3-4 loop (alpha1-(309-392)) of the human GlyR alpha1-subunit were individually expressed in HEK293 cells and in Escherichia coli. The extracellular domain obtained from E. coli expression was purified in its denatured form and refolding conditions were established. Circular dichroism and Fourier-transform-infrared spectroscopy suggested approximately 25% alpha-helix and approximately 48% beta-sheet for the extracellular domain, while no alpha-helices were detectable for the TM3-4 loop. Size exclusion chromatography and sucrose density centrifugation indicated that isolated glycine receptor domains assembled into multimers of distinct molecular weight. For the extracellular domain from E. coli, we found an apparent molecular weight compatible with a 15mer by gel filtration. The N-terminal domain from HEK293 cells, analyzed by sucrose gradient centrifugation, showed a bimodal distribution, suggesting oligomerization of approximately 5 and 15 subunits. Likewise, for the intracellular domain from E. coli, a single molecular mass peak of approximately 49 kDa indicated oligomerization in a defined native structure. As shown by [(3)H]strychnine binding, expression in HEK293 cells and refolding of the isolated extracellular domain reconstituted high affinity antagonist binding. Cell fractionation, alkaline extraction experiments, and immunocytochemistry showed a tight plasma membrane association of the isolated GlyR N-terminal protein. These findings indicate that distinct functional characteristics of the full-length GlyR are retained in the isolated N-terminal domain.     

The N-terminal domains of acetylcholine receptor subunits contain recognition signals for the initial steps of receptor assembly.

Ligand-gated ion channels are oligomeric membrane proteins in which homologous subunits specifically recognize one another and assemble around an aqueous pore. To identify domains responsible for the specificity of subunit association, we used a dominant-negative assay in which truncated subunits of the mouse muscle acetylcholine receptor (AChR) were coexpressed with the four wild-type subunits in transfected COS cells. Fragments of the alpha, delta, and gamma subunits consisting solely of the extracellular N-terminal domain blocked surface expression of the AChR and the formation of alpha delta heterodimers, an early step in the assembly pathway of the AChR. Immunoprecipitation and sucrose gradient sedimentation experiments showed that an N-terminal fragment of the alpha subunit forms a specific complex with the intact delta subunit. Thus the extracellular N-terminal domain of the alpha, delta, and gamma subunits contains the information necessary for specific subunit association.     

Differential regulation of corticotropin releasing factor 1alpha receptor endocytosis and trafficking by beta-arrestins and Rab GTPases

The corticotropin releasing factor (CRF) type 1alpha receptor, a member of the G protein-coupled receptor (GPCR) subfamily B, is involved in the aetiology of anxiety and depressive disorders. In the present study, we examined the internalization and trafficking of the CRF1alpha receptor in both human embryonic kidney (HEK)293 cells and primary cortical neurons. We found that CRF1alpha receptor activation leads to the selective recruitment of beta-arrestin2 in both HEK293 cells and neurons. We observed distinct distribution patterns of CRF1alpha receptor and beta-arrestin2 in HEK293 cells and cortical neurons. In HEK293 cells, beta-arrestin2-green fluorescent protein (GFP) co-localized with CRF1alpha receptor in vesicles at the plasma membrane but was dissociated from the receptor in endosomes. In contrast, in primary cortical neurons, beta-arrestin2 and CRF1alpha receptor were internalized in distinct endocytic vesicles. By bioluminescence resonance energy transfer, we demonstrated that beta-arrestin2 association with CRF1alpha receptor was increased in cells transfected with G protein-coupled receptor kinase (GRK)3 and GRK6 and decreased in cells transfected with GRK2 and GRK5. In both HEK293 cells and cortical neurons, internalized CRF1alpha receptor transited from Rab5-positive early endosomes to Rab4-positive recycling endosomes and was not targeted to lysosomes. However, CRF1alpha receptor resensitization was blocked by the overexpression of wild-type, but not dominant-negative, Rab5 and Rab4 GTPases. Taken together, our results suggest that beta-arrestin trafficking differs between HEK293 cells and neurons, and that CRF1alpha receptor resensitization is regulated in an atypical manner by Rab GTPases.     

Cell surface expression of functional hepatitis C virus E1 and E2 glycoproteins

Hepatitis C virus (HCV) glycoproteins E1 and E2 are believed to be retained in the endoplasmic reticulum (ER) or cis-Golgi compartment via retention signals located in their transmembrane domains. Here we describe the detection of E1 and E2 at the surface of transiently transfected HEK 293T and Huh7 cells. Surface-localized E1E2 heterodimers presented exclusively as non-covalently associated complexes. Surface-expressed E2 contained trans-Golgi modified complex/hybrid type carbohydrate and migrated diffusely between 70 and 90 kDa while intracellular E1 and E2 existed as high mannose 35 kDa and 70 kDa precursors, respectively. In addition, surface-localized E1E2 heterodimers were incorporated into E1E2-pseudotyped HIV-1 particles that were competent for entry into Huh7 cells. These studies suggest that functional HCV glycoproteins are not retained exclusively in the ER and transit through the secretory pathway.     

Characterization of an N-terminal secreted domain of the type-1 human metabotropic glutamate receptor produced by a mammalian cell line

A Chinese hamster ovary cell line has been established which secretes the N-terminal domain of human mGlu1 receptor. The secreted protein has been modified to contain a C-terminal hexa-histidine tag and can be purified by metal-chelate chromatography to yield a protein with an apparent molecular weight of 130 kDa. Following treatment with dithiothreitol the apparent molecular weight is reduced to 75 kDa showing that the protein is a disulphide-bonded dimer. N-terminal protein sequencing of both the reduced and unreduced forms of the protein yielded identical sequences, confirming that they were derived from the same protein, and identifying the site of signal-peptide cleavage of the receptor as residue 32 in the predicted amino acid sequence. Endoglycosidase treatment of the secreted and intracellular forms of the protein showed that the latter was present as an endoglycosidase H-sensitive dimer, indicating that dimerization is taking place in the endoplasmic reticulum. Characterization of the binding of [3H]quisqualic acid showed that the protein was secreted at levels of up to 2.4 pmol/mL and the secreted protein has a Kd of 5.6 +/- 1.8 nm compared with 10 +/- 1 nm for baby hamster kidney (BHK)-mGlu1alpha receptor-expressing cell membranes. The secreted protein maintained a pharmacological profile similar to that of the native receptor and the binding of glutamate and quisqualate were unaffected by changes in Ca2+ concentration.     

Heterodimerization of calcium sensing receptors with metabotropic glutamate receptors in neurons

Calcium sensing (CaR) and Group I metabotropic glutamate receptors exhibit overlapping expression patterns in brain, and share common signal transduction pathways. To determine whether CaR and Group I metabotropic glutamate receptors (mGluRs) (mGluR1alpha and mGluR5) can form heterodimers, we immunoprecipitated CaR from bovine brain and observed co-precipitation of mGluR1alpha. CaR and mGluR1alpha co-localize in hippocampal and cerebellar neurons, but are expressed separately in other brain regions. In vitro transfection studies in HEK-293 cells established the specificity and disulfide-linked nature of the CaR:mGluR1alpha (CaR:mGluR5) interactions. CaR:mGluR1alpha (CaR:mGluR5) heterodimers exhibit altered trafficking via Homer 1c when compared with CaR:CaR homodimers. CaR becomes sensitive to glutamate-mediated internalization when present in CaR:mGluR1alpha heterodimers. These results demonstrate cross-family covalent heterodimerization of CaR with Group I mGluRs, and increase the potential role(s) for CaR in modulating neuronal function.     

Interaction of integrin-linked kinase with the kidney chloride/bicarbonate exchanger, kAE1

Kidney anion exchanger 1 (kAE1) mediates chloride/bicarbonate exchange at the basolateral membrane of kidney alpha-intercalated cells, thereby facilitating bicarbonate reabsorption into the blood. Human kAE1 lacks the N-terminal 65 residues of the erythroid form (AE1, band 3), which are essential for binding of cytoskeletal and cytosolic proteins. Yeast two-hybrid screening identified integrin-linked kinase (ILK), a serine/threonine kinase, and an actin-binding protein as an interacting partner with the N-terminal domain of kAE1. Interaction between kAE1 and ILK was confirmed in co-expression experiments in HEK 293 cells and is mediated by a previously unidentified calponin homology domain in the kAE1 N-terminal region. The calponin homology domain of kAE1 binds the C-terminal catalytic domain of ILK to enhance association of kAE1 with the actin cytoskeleton. Overexpression of ILK increased kAE1 levels at the cell surface as shown by flow cytometry, cell surface biotinylation, and anion transport activity assays. Pulse-chase experiments revealed that ILK associates with kAE1 early in biosynthesis, likely in the endoplasmic reticulum. ILK co-localized with kAE1 at the basolateral membrane of polarized Madin-Darby canine kidney cells and in alpha-intercalated cells of human kidneys. Taken together these results suggest that ILK and kAE1 traffic together from the endoplasmic reticulum to the basolateral membrane. ILK may provide a linkage between kAE1 and the underlying actin cytoskeleton to stabilize kAE1 at the basolateral membrane, resulting in higher levels of cell surface expression.     

CD4同源二聚化或寡聚化的间接检测及其介导的生物学功能的初步分析

近年来的研究表明 ,CD4分子于细胞膜表面不仅以单体形式存在还可以通过其D4和D1形成同源二聚化及寡聚化 ,并且二聚化及寡聚化的CD4分子才能稳定地与MHC Ⅱ类分子结合。通过分析CD4分子以及CD4缺失突变体分子融合Fas基因片段所诱导的转染靶细胞的凋亡 ,以及携带绿色荧光蛋白的CD4分子转染HEK2 93细胞所筛选出的稳定克隆的不同荧光强度和与MHC Ⅱ类分子阳性细胞Raji之间的不同黏附效应间接鉴定CD4同源二聚体或寡聚体的存在 ,并对二聚化或寡聚化CD4分子所介导的生物学功能进行初步分析。     

Cell-type-specific pathways of neurotensin endocytosis

The neurotensin receptor subtype 1 (NTS1) is a G-protein-coupled receptor (GPCR) mediating a large number of central and peripheral effects of neurotensin. Upon stimulation, NTS1 is rapidly internalized and targeted to lysosomes. This process depends on the interaction of the phosphorylated receptor with β–arrestin. Little is known about other accessory endocytic proteins potentially involved. Here, we investigated the involvement of dynamin, amphiphysin, and intersectin in the internalization of NTS1 receptor-ligand complexes in transfected COS-7 and HEK 293 cells, by using the transferrin receptor as an internal control for the constitutive endocytic pathway. We found that NTS1 endocytosis was not only arrestin–dependent, but also dynamin–dependent in both COS-7 and HEK 293 cells, whereas internalization of the transferrin receptor was independent of arrestin but required dynamin. Overexpression of the SH3 domain of amphiphysin II had no effect on receptor internalization in either cell type. By contrast, overexpression of full-length intersectin or of its SH3 domain (but not of its EH domain) inhibited NTS1 internalization in COS-7 but not in HEK 293 cells. This difference between COS-7 and HEK 293 cells was not attributable to differences in endogenous intersectin levels between the two cell lines. Indeed, the same constructs inhibited transferrin endocytosis equally well in COS-7 and HEK 293 cells. However, immunogold electron microscopy revealed that internalized NTS1 receptors were associated with clathrin-coated pits in COS-7 cells but with smooth vesicles in HEK 293 cells, suggesting that NTS1 internalization proceeds via different endocytic pathways in these two cell types. This work was supported by grants to A.B. from CIHR and FRSQ.     

A novel MaxiK splice variant exhibits dominant-negative properties for surface expression

We identified a novel MaxiK alpha subunit splice variant (SV1) from rat myometrium that is also present in brain. SV1 has a 33-amino acid insert in the S1 transmembrane domain that does not alter S1 overall hydrophobicity, but makes the S0-S1 linker longer. SV1 was transfected in HEK293T cells and studied using immunocytochemistry and electrophysiology. In non-permeabilized cells, N-terminal c-Myc- or C-terminal green fluorescent protein-tagged SV1 displayed no surface labeling or currents. The lack of SV1 functional expression was due to endoplasmic reticulum (ER) retention as determined by colabeling experiments with a specific ER marker. To explore the functional role of SV1, we coexpressed SV1 with the alpha (human SLO) and beta1 (KCNMB1) subunits of the MaxiK channel. Coexpression of SV1 inhibited surface expression of alpha and beta1 subunits approximately 80% by trapping them in the ER. This inhibition seems to be specific for MaxiK channel subunits since SV1 was unable to prevent surface expression of the Kv4.3 channel or to interact with green fluorescent protein. These results indicate a dominant-negative role of SV1 in MaxiK channel expression. Moreover, they reveal down-regulation by splice variants as a new mechanism that may contribute to the diverse levels of MaxiK channel expression in non-excitable and excitable cells.     

Syntrophins regulate alpha1D-adrenergic receptors through a PDZ domain-mediated interaction

To find novel cytoplasmic binding partners of the alpha1D-adrenergic receptor (AR), a yeast two-hybrid screen using the alpha1D-AR C terminus as bait was performed on a human brain cDNA library. Alpha-syntrophin, a protein containing one PDZ domain and two pleckstrin homology domains, was isolated in this screen as an alpha1D-AR-interacting protein. Alpha-syntrophin specifically recognized the C terminus of alpha1D- but not alpha1A- or alpha1B-ARs. In blot overlay assays, the PDZ domains of syntrophin isoforms alpha, beta1, and beta2 but not gamma1 or gamma2 showed strong selective interactions with the alpha1D-AR C-tail fusion protein. In transfected human embryonic kidney 293 cells, full-length alpha1D- but not alpha1A- or alpha1B-ARs co-immunoprecipitated with syntrophins, and the importance of the receptor C terminus for the alpha1D-AR/syntrophin interaction was confirmed using chimeric receptors. Mutation of the PDZ-interacting motif at the alpha1D-AR C terminus markedly decreased inositol phosphate formation stimulated by norepinephrine but not carbachol in transfected HEK293 cells. This mutation also dramatically decreased alpha1D-AR binding and protein expression. In addition, stable overexpression of alpha-syntrophin significantly increased alpha1D-AR protein expression and binding but did not affect those with a mutated PDZ-interacting motif, suggesting that syntrophin plays an important role in maintaining receptor stability by directly interacting with the receptor PDZ-interacting motif. This direct interaction may provide new information about the regulation of alpha1D-AR signaling and the role of syntrophins in modulating G protein-coupled receptor function.     

The extracellular N-terminal domain and transmembrane domains 1 and 2 mediate oligomerization of a yeast G protein-coupled receptor

G protein-coupled receptors (GPCRs) can form homodimers/oligomers and/or heterodimers/oligomers. The mechanisms used to form specific GPCR oligomers are poorly understood because the domains that mediate such interactions and the step(s) in the secretory pathway where oligomerization occurs have not been well characterized. Here we have used subcellular fractionation and fluorescence resonance energy transfer (FRET) experiments to show that oligomerization of a GPCR (alpha-factor receptor; STE2 gene product) of the yeast Saccharomyces cerevisiae occurs in the endoplasmic reticulum. To identify domains of this receptor that mediate oligomerization, we used FRET and endocytosis assays of oligomerization in vivo to analyze receptor deletion mutants. A mutant lacking the N-terminal extracellular domain and transmembrane (TM) domain 1 was expressed at the cell surface but did not self-associate. In contrast, a receptor fragment containing only the N-terminal extracellular domain and TM1 could self-associate and heterodimerize with wild type receptors. Analysis of other mutants suggested that oligomerization is facilitated by the N-terminal extracellular domain and TM2. Therefore, the N-terminal extracellular domain, TM1, and TM2 appear to stabilize alpha-factor receptor oligomers. These domains may form an interface in contact or domain-swapped oligomers. Similar domains may mediate dimerization of certain mammalian GPCRs.