Transmembrane region of N-methyl-D-aspartate receptor (NMDAR) subunit is required for receptor subunit assembly

N-Methyl-D-aspartate receptors (NMDARs), one of three main classes of ionotropic glutamate receptors, play major roles in synaptic plasticity, synaptogenesis, and excitotoxicity. Unlike non-NMDA receptors, NMDARs are thought to comprise obligatory heterotetrameric complexes mainly composed of GluN1 and GluN2 subunits. When expressed alone in heterogenous cells, such as HEK293 cells, most of the NMDAR subunits can neither leave the endoplasmic reticulum (ER) nor be expressed in the cell membrane because of the ER retention signals. Only when NMDARs are heteromerically assembled can the ER retention signals be masked and NMDARs be expressed in the surface membrane. However, the mechanisms underlying NMDAR assembly remain poorly understood. To identify regions in subunits that mediate this assembly, we made a series of truncated or chimeric cDNA constructs. Using FRET measurement in living cells combined with immunostaining and coimmunoprecipitation analysis, we examined the assembly-determining domains of NMDAR subunits. Our results indicate that the transmembrane region of subunits is necessary for the assembly of NMDAR subunits, both for the homodimer and the heteromer.     

Correct identification of the chloroplastic protoporphyrinogen IX oxidase N-terminus places the biochemical data in frame

CTLA-4 gene constructs were designed to express CTLA-4 exclusively in the endoplasmic reticulum (ER). Four different CTLA-4 gene constructs were transfected into HEK 293 (human embryonic kidney) and A20 (Balb/c mouse B lymphoma) cells. All constructs contained an ER retention signal and coded for CTLA-4 expression in the ER. One of the constructs, which contained the membrane part of CTLA-4, coded for an expression both on the cell surface and in the ER. Three of the expressed CTLA-4 types (including the ER-membrane-expressed form) caused a reduced surface expression of B7 in the A20 cells. Only constructs which allow dimerization of CTLA-4 showed this effect. It is assumed that intracellular CTLA-4 bound B7 and inhibited therefore the transport of B7 to the surface. The binding obviously caused also an enhanced degradation of the complexes because both proteins showed a low concentration in the transfected cell lines. CTLA-4-transfected and B7-reduced A20 cells showed a diminished costimulating activity upon T cells. This was demonstrated by a reduced proliferation of T cells from ovalbumin-immunized Balb/c mice, incubated with ovalbumin peptide-primed CTLA-4-transfected A20 cells.     

An Endoplasmic Reticulum Retention Signal Located in the Extracellular Amino-terminal Domain of the NR2A Subunit of N-Methyl-d-aspartate Receptors

N-Methyl-d-aspartate (NMDA) receptors play critical roles in complex brain functions as well as pathogenesis of neurodegenerative diseases. There are many NMDA isoforms and subunit types that, together with subtype-specific assembly, give rise to significant functional heterogeneity of NMDA receptors. Conventional NMDA receptors are obligatory heterotetramers composed of two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits. When individually expressed in heterogeneous cells, most of the NR1 splice variants and the NR2 subunits remain in the endoplasmic reticulum (ER) and do not form homomeric channels. The mechanisms underlying NMDA receptor trafficking and functional expression remain uncertain. Using truncated and chimeric NMDA receptor subunits expressed in heterogeneous cells and hippocampal neurons, together with immunostaining, biochemical, and functional analyses, we found that the NR2A amino-terminal domain (ATD) contains an ER retention signal, which can be specifically masked by the NR1a ATD. Interestingly, no such signal was found in the ATD of the NR2B subunit. We further identified the A2 segment of the NR2A ATD to be the primary determinant of ER retention. These findings indicate that NR2A-containing NMDA receptors may undergo a different ER quality control process from NR2B-containing NMDA receptors.Ionotropic glutamate receptors (iGluRs)² mediate most of the excitatory neurotransmission in the central nervous system. They play key roles in complex brain functions as well as in the pathogenesis of neurodegenerative diseases. Based on pharmacological properties and sequence similarities, iGluRs can be grouped into three major subtypes: GluR1 to -4 subunits form α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, GluR5 to -7 and KA1 and -2 subunits make up kainate receptors, and NR1 together with NR2A to -D subunits comprise the NMDA receptors (1). All iGluR subunits share a unique membrane topology with a large extracellular NH₂-terminal domain, three transmembrane segments (TM1 (transmembrane domain 1), TM3, and TM4), a P-loop region, and a cytoplasmic COOH terminus (2, 3). Based on the sequence homology to bacterial periplasmic binding proteins, the NH₂-terminal domain of iGluRs can be divided into two domains in tandem: the amino-terminal domain (ATD), which includes the first 400 or so amino acids (4), and the following S1 domain preceding TM1, which forms the ligand-binding domain together with the extracellular loop between TM3 and TM4 (S2 domain) (5, 6).Among iGluRs, NMDA receptors are special in that conventional NMDA receptors are obligatory tetrameric membrane proteins composed of two glycine-binding NR1 and two glutamate-binding NR2 subunits. The NR1 subunit is essential for the formation of functional NMDA receptor channel, whereas the NR2 subunit modifies channel properties, such as current kinetics and channel conductance (1). The major NR1 splice variant and the NR2 subunits are retained in the ER when expressed alone in heterogeneous cells. Only when expressed together do they form functional receptors on the cell surface (7–9). In the last decade, enormous progress has been made in understanding the phenomenology and mechanisms of functional plasticity of NMDA receptors. However, much less is known about the mechanisms underlying the ER retention of NMDA receptor subunits. Previous studies focused on the COOH terminus have shown that the NR1a subunit contains an ER retention signal, RRR, in the C1 cassette, whereas a motif, HLFY, found in the NR2B subunit immediately following the TM4 (10) or, at least, the presence of any two amino acid residues after NR2 TM4 (11) is required for the export of NR1-NR2 complexes from the ER. Recently, novel ER retention signals were identified in the TM3 of both NR1 and NR2B subunits. In addition, TM3 of both NR1 and NR2B and TM4 of NR1 are necessary for masking ER retention signals found in TM3 (12).In the present study, we focused on the functional role of the ATD in the surface expression of NMDA receptors. Interestingly, we found an ER retention signal located in the ATD of the NR2A subunit but not in the corresponding domain of the NR2B. It is suggested that NR2A-containing NMDA receptors may undergo an ER quality control process different from that of NR2B-containing NMDA receptors.     

A trafficking checkpoint controls GABA(B) receptor heterodimerization

Surface expression of GABA(B) receptors requires heterodimerization of GB1 and GB2 subunits, but little is known about mechanisms that ensure efficient heterodimer assembly. We found that expression of the GB1 subunit on the cell surface is prevented through a C-terminal retention motif RXR(R); this sequence is reminiscent of the ER retention/retrieval motif RKR identified in subunits of the ATP-sensitive K+ channel. Interaction of GB1 and GB2 through their C-terminal coiled-coil alpha helices masks the retention signal in GB1, allowing the plasma membrane expression of the assembled complexes. Because individual GABA(B) receptor subunits and improperly assembled receptor complexes are not functional even if expressed on the cell surface, we conclude that a trafficking checkpoint ensures efficient assembly of functional GABA(B) receptors.     

RIC-3 Exclusively Enhances the Surface Expression of Human Homomeric 5-Hydroxytryptamine Type 3A (5-HT3A) Receptors Despite Direct Interactions with 5-HT3A, -C, -D, and -E Subunits

Although five 5-hydroxytryptamine type 3 (5-HT3) subunits (A–E) have been cloned, knowledge on the regulation of their assembly is limited. RIC-3 has been identified as a chaperone specific for the pentameric ligand-gated nicotinic acetylcholine and 5-HT₃ receptors. Therefore, we examined the impact of RIC-3 on differently composed 5-HT₃ receptors with the focus on 5-HT3C, -D, and -E subunits. The influence of RIC-3 on these receptor subtypes is supported by the presence of RIC3 mRNA in tissues expressing at least one of the subunits 5-HT3C, -D, and -E. Furthermore, immunocytochemical studies on transfected mammalian cells revealed co-localization in the endoplasmic reticulum and direct interaction of RIC-3 with 5-HT3A, -C, -D, and -E. Functional and pharmacological characterization was performed using HEK293 cells expressing 5-HT3A or 5-HT3A + 5-HT3B (or -C, -D, or -E) in the presence or absence of RIC-3. Ca²⁺ influx analyses revealed that RIC-3 does not influence the 5-HT concentration-response relationship on 5-HT₃A receptors but leads to differential increases of 5-HT-induced maximum response (E_max) on cells expressing different subunits. Increases of E_max were due to analogously enhanced B_max values for binding of the 5-HT₃ receptor antagonist [³H]GR65630. The observed enhanced cell surface expression of the tested 5-HT3 subunit combinations correlated with the increased surface expression of 5-HT3A as determined by flow cytometry. In conclusion, we showed that RIC-3 can interact with 5-HT3A, -C, -D, and -E subunits and predominantly enhances the surface expression of homomeric 5-HT₃A receptors in HEK293 cells. These data implicate a possible role of RIC-3 in determining 5-HT₃ receptor composition in vivo.     

Role of the transmembrane and extracytoplasmic domain of beta subunits in subunit assembly, intracellular transport, and functional expression of Na,K-pumps

The ubiquitous Na,K- and the gastric H,K-pumps are heterodimeric plasma membrane proteins composed of an alpha and a beta subunit. The H,K- ATPase beta subunit (beta HK) can partially act as a surrogate for the Na,K-ATPase beta subunit (beta NK) in the formation of functional Na,K- pumps (Horisberger et al., 1991. J. Biol. Chem. 257:10338-10343). We have examined the role of the transmembrane and/or the ectodomain of beta NK in (a) its ER retention in the absence of concomitant synthesis of Na,K-ATPase alpha subunits (alpha NK) and (b) the functional expression of Na,K-pumps at the cell surface and their activation by external K+. We have constructed chimeric proteins between Xenopus beta NK and rabbit beta HK by exchanging their NH2-terminal plus transmembrane domain with their COOH-terminal ectodomain (beta NK/HK, beta HK/NK). We have expressed these constructs with or without coexpression of alpha NK in the Xenopus oocyte. In the absence of alpha NK, Xenopus beta NK and all chimera that contained the ectodomain of beta NK were retained in the ER while beta HK and all chimera with the ectodomain of beta HK could leave the ER suggesting that ER retention of unassembled Xenopus beta NK is mediated by a retention signal in the ectodomain. When coexpressed with alpha NK, only beta NK and beta NK/HK chimera assembled efficiently with alpha NK leading to similar high expression of functional Na,K-pumps at the cell surface that exhibited, however, a different apparent K+ affinity. beta HK or chimera with the transmembrane domain of beta HK assembled less efficiently with alpha NK leading to lower expression of functional Na,K-pumps with a different apparent K+ affinity. The data indicate that the transmembrane domain of beta NK is important for efficient assembly with alpha NK and that both the transmembrane and the ectodomain of beta subunits play a role in modulating the transport activity of Na,K- pumps.     

The identification of N-glycosylated residues of the human 5-HT3B receptor subunit: importance for cell membrane expression

The 5-hydroxytryptamine 3 (5-HT(3)) receptor is a pentameric ligand-gated ion channel with potential molecular isoforms arising from different subunit combinations and/or different post-translational modifications of the individual subunits. Since N-glycosylation of the 5-HT3A subunit impacts cell surface trafficking, the presence of N-glycosylation of the human (h) 5-HT3B subunit and the influence upon cell membrane expression was investigated. Following transient expression of the h5-HT3B subunit by human embryonic kidney cells (HEK293 cells) stably expressing the h5-HT3A subunit, the N-glycosylation inhibitor tunicamycin reduced the size of the predominant h5-HT3B-immunoreactive protein (～ 55 kDa reduced to ～ 40 kDa). Disruption of each consensus N-glycosylation sequences in the h5-HT3B subunit (N31S, N75S, N117S, N147S and N182S) resulted in a reduced molecular weight (by ～ 2-4 kDa) of each mutant when expressed by HEK293 cells stably expressing the h5-HT3A subunit. Immunocytochemical studies demonstrated that disruption of each of the N-glycosylation sequences (individually or combined) reduced the expression of the mutant h5-HT3B subunit protein in the cell membrane when co-expressed with the h5-HT3A subunit. The present study has identified utilised N-glycosylation sites of the h5-HT3B subunit and demonstrated that they promote subunit expression in the cell membrane; a prerequisite for 5-HT(3) receptor function.     

Structural and functional dissection of human cytomegalovirus US3 in binding major histocompatibility complex class I molecules

The human cytomegalovirus US3, an endoplasmic reticulum (ER)-resident transmembrane glycoprotein, forms a complex with major histocompatibility complex (MHC) class I molecules and retains them in the ER, thereby preventing cytolysis by cytotoxic T lymphocytes. To identify which parts of US3 confine the protein to the ER and which parts are responsible for the association with MHC class I molecules, we constructed truncated mutant and chimeric forms in which US3 domains were exchanged with corresponding domains of CD4 and analyzed them for their intracellular localization and the ability to associate with MHC class I molecules. All of the truncated mutant and chimeric proteins containing the luminal domain of US3 were retained in the ER, while replacement of the US3 luminal domain with that of CD4 led to cell surface expression of the chimera. Thus, the luminal domain of US3 was sufficient for ER retention. Immunolocalization of the US3 glycoprotein after nocodazole treatment and the observation that the carbohydrate moiety of the US3 glycoprotein was not modified by Golgi enzymes indicated that the ER localization of US3 involved true retention, without recycling through the Golgi. Unlike the ER retention signal, the ability to associate with MHC class I molecules required the transmembrane domain in addition to the luminal domain of US3. Direct interaction between US3 and MHC class I molecules could be demonstrated after in vitro translation by coimmunoprecipitation. Together, the present data indicate that the properties that allow US3 to be localized in the ER and bind MHC class I molecules are located in different parts of the molecule.     

Differential subcellular localization of RIC-3 isoforms and their role in determining 5-HT3 receptor composition

RIC-3 has been identified as a chaperone molecule involved in promoting the functional expression of nicotinic acetylcholine and 5-HT(3) receptors in mammalian cells. In this study, we examined the effects of RIC-3a (isoform a) and a truncated isoform (isoform d) on RIC-3 localization, mobility, and aggregation and its effect on 5-HT3 receptor composition in mammalian cells. Human RIC-3a possesses an amino-terminal signal sequence that targets it to the endoplasmic reticulum where it is distributed within the reticular network, often forming large diffuse "slicks" and bright "halo" structures. RIC-3a is highly mobile within and between these compartments. Despite the propensity for RIC-3a to aggregate, its expression enhances the level of surface 5-HT3A (homomeric) receptors. In contrast, RIC-3a exerts an inhibitory action on the surface expression of heteromeric 5-HT3A/B receptors. RIC-3d exhibits an altered subcellular distribution, being localized to the endoplasmic reticulum, large diffuse slicks, tubulo-vesicular structures, and the Golgi. Bidirectional trafficking between the endoplasmic reticulum and Golgi suggests that RIC-3d constitutively cycles between these two compartments. In support of the large coiled-coil domain of RIC-3a being responsible for protein aggregation, RIC-3d, lacking this cytoplasmic domain, does not aggregate or induce the formation of bright aggregates. Regardless of these differences, isoform d is still capable of enhancing homomeric, and inhibiting heteromeric, 5-HT3 receptor expression. Thus, both isoforms of RIC-3 play a role in determining 5-HT3 receptor composition.     

Export from the endoplasmic reticulum of assembled N-methyl-d-aspartic acid receptors is controlled by a motif in the c terminus of the NR2 subunit

Functional N-methyl-d-aspartic acid (NMDA) receptors are formed from the assembly of NR1 and NR2 subunits. When expressed alone, the major NR1 splice variant and the NR2 subunits are retained in the endoplasmic reticulum (ER), reflecting a quality control mechanism found in many complex multisubunit proteins to ensure that only fully assembled and properly folded complexes reach the cell surface. Recent studies have identified an RRR motif in the C terminus of the NR1 subunit, which controls the ER retention of the unassembled subunit. Here we investigated the mechanisms controlling the ER retention of the NR2 subunit and the export of the assembled complex from the ER. We found that Tac chimeras of the C terminus of the NR2B subunit show that an ER retention signal is also present in the NR2B subunit. In assembled complexes, ER retention signals on the individual subunits must be overcome to allow the complex to leave the ER. One common mechanism involves mutual masking of the signals on the individual subunits. Our data do not support such a mechanism for regulating the release of assembled NMDA receptors from the ER. We found that the motif, HLFY, immediately following transmembrane domain 4 of the NR2 subunit, is required for the assembled complex to exit from the ER. Mutation of this motif allowed the assembly of NR1 and NR2 subunits into a complex that was functional, based on MK-801 binding, but it is retained in the ER. These results are consistent with HLFY functioning as a signal that is necessary for the release of the assembled functional NMDA receptor complex from the ER.     

Endoplasmic reticulum localization of Gaa1 and PIG-T, subunits of the glycosylphosphatidylinositol transamidase complex

After integration into the endoplasmic reticulum (ER) membrane, ER-resident membrane proteins must be segregated from proteins that are exported to post-ER compartments. Here we analyze how human Gaa1 and PIG-T, two of the five subunits of the ER-localized glycosylphosphatidylinositol transamidase complex, are retained in the ER. Neither protein contains a known ER localization signal. Gaa1 is a polytopic membrane glycoprotein with a cytoplasmic N terminus and a large luminal loop between its first two transmembrane spans; PIG-T is a type I membrane glycoprotein. To simplify our analyses, we studied Gaa1 and PIG-T constructs that could not interact with other subunits of the transamidase. We now show that Gaa1(282), a truncated protein consisting of the first TM domain and luminal loop of Gaa1, is correctly oriented, N-glycosylated, and ER-localized. Removal of a potential ER localization signal in the form of a triple arginine cluster near the N terminus of Gaa1 or Gaa1(282) had no effect on ER localization. Fusion proteins consisting of different elements of Gaa1(282) appended to alpha2,6-sialyltransferase or transferrin receptor could exit the ER, indicating that Gaa1(282), and by implication Gaa1, does not contain any dominant ER-sorting determinants. The data suggest that Gaa1 is passively retained in the ER by a signalless mechanism. In contrast, similar analyses of PIG-T revealed that it is ER-localized because of information in its transmembrane span; fusion of the PIG-T transmembrane span to Tac antigen, a plasma membrane-localized protein, caused the fusion protein to remain in the ER. These data are discussed in the context of models that have been proposed to account for retention of ER membrane proteins.     

Protein targeting to endoplasmic reticulum by dilysine signals involves direct retention in addition to retrieval.

Dilysine signals confer localization of type I membrane proteins to the endoplasmic reticulum (ER). According to the prevailing model these signals target proteins to the ER by COP I-mediated retrieval from post-ER compartments, whereas the actual retention mechanism in the ER is unknown. We expressed chimeric membrane proteins with a C-terminal -Lys-Lys-Ala-Ala (KKAA) or -Lys-Lys-Phe-Phe (KKFF) dilysine signal in Lec-1 cells. Unlike KKFF constructs, which had access to post-ER compartments, the KKAA chimeras were localized to the ER by confocal microscopy and were neither processed by cis-Golgi-specific enzymes in vivo nor included into ER-derived transport vesicles in an in vitro budding assay, suggesting that KKAA-bearing proteins are permanently retained in the ER. The ER localization was nonsaturable and exclusively mediated by the dilysine signal because mutating the lysines to alanines led to cell surface expression of the chimeras. Although the KKAA signal avidly binds COP I in vitro, the ER retention by this signal does not depend on intact COP I in vivo because it was not affected in an epsilon-COP-deficient cell line. We propose that dilysine ER targeting signals can mediate ER retention in addition to retrieval.     

Masking of an endoplasmic reticulum retention signal by its presence in the two subunits of the asialoglycoprotein receptor

Human asialoglycoprotein receptor H1 and H2b subunits assemble into a hetero-oligomer that travels to the cell surface. The H2a variant on the other hand is a precursor of a cleaved soluble form that is secreted. Uncleaved H2a precursor molecules cannot exit the endoplasmic reticulum (ER), a lumenal juxtamembrane pentapeptide being responsible for their retention. Insertion of this pentapeptide into H1 (H1i5) causes its complete ER retention but not fast degradation as happens to H2a. Cotransfection of H2a elicited, by heterodimerization, the Golgi processing of H1i5 and its surface expression. This occurred to a much lesser extent by cotransfection of H2b. Likewise, coexpression of H1i5 and not H1 stabilized H2a and caused its export to the cell surface. Homodimerization of molecules containing the pentapeptide did not cancel the retention. Thus, only when the pentapeptide is present in both subunits is the ER retention efficiently abrogated. The results show the unexpected finding that identical ER retention signals present in two associated chains can mask and cancel each other's effect. This could have important implications as similar abrogation of ER retention of other proteins could eventually be obtained by engineering and coexpressing an associated protein containing the same retention signal.     

Differential Expression of the 5-HT3A and 5-HT3B Receptor in Differentiated NG108-15 Cells

Previous work from this laboratory has shown that the serotonin (5-HT) induced response is significantly augmented in differentiated NG108-15 (NG) cells treated with dibutyryl cAMP (Bt(2)cAMP) due to qualitative and quantitative changes in the expression of the 5-HT(3) receptor as demonstrated by specific [(3)H] LY-278584 (a selective 5HT(3) receptor antagonist) binding. In this study, we investigated whether there is any change in the relative expression of the 5-HT(3A) and 5-HT(3B) subunits in NG cells differentiated following Bt(2)cAMP treatment cells. The major findings of this study were that the relative amount of 5-HT(3B) subunit mRNA in Bt(2)cAMP-treated NG cells 5 days following Bt(2)cAMP-treatment was greater than that in the untreated cells. In contrast, the relative expression of the 5-HT(3B) subunit protein in the Bt(2)cAMP-treated NG cells was much less than in the untreated cells, but the relative expression of the 5-HT(3A) subunit in the Bt(2)cAMP-treated NG cells was similar to the untreated cells. Therefore, no relationship between mRNA and protein expression for 5-HT(3A) and 5-HT(3B) subunits in Bt(2)cAMP treated and untreated NG cells were observed. It was also found that fluorescent intensity for the 5-HT(3B) subunit in the cell body of the Bt(2)cAMP treated and untreated NG cells gradually decreased from the day 1-5 after Bt(2)cAMP treatment. However, in specific areas such as the varicosity and nerve endings of the Bt(2)cAMP treated cells, staining intensity for the 5-HT(3B) subunits was stronger than in the untreated cells at the all time points, peaking at day 5 post-treatment. These results suggest that the augmented response induced by 5-HT acting via 5-HT(3) receptors in differentiated NG cells may be due to changes in the relative amount of the 5-HT(3B) subunit, particularly the ratio and distribution of the 5-HT(3A) to (3B) subunits.     

Role of the fourth membrane domain of the NR2B subunit in the assembly of the NMDA receptor

N-methyl-D-aspartate (NMDA) receptors play crucial roles in excitatory synaptic transmission as well as in excitotoxicity. A growing body of evidence suggests that the regulation of both subunit composition and the number of NMDA receptors reaching the surface membrane are tightly regulated. Recently, we have shown that the third membrane domains (M3) of both NR1 and NR2B subunits contain endoplasmic reticulum (ER) retention signals that prevent the unassembled subunits from leaving the ER. Furthermore, these membrane domains together with NR1 M4 are necessary for negating the ER retention signals found in M3 of NR1 and NR2B. In this addendum, we present new electrophysiological data showing that mutation of the HLFY motif, located immediately after M4 of the NR2B subunit, abolishes the surface trafficking of full-length NR1/NR2B complexes (supporting previous immunofluorescent experiments from our lab); however, the deletion of the NR2B C-terminus including the HLFY motif did not affect the formation of functional receptors when two pieces of the NR2B subunit, NR2B truncated before M4 and NR2B M4, were co-expressed together with the NR1 subunit. These observations will help to uncover the processes involved in the assembly of NR1 and NR2 subunits into functional NMDA receptors.     

Role of the fourth membrane domain of the NR2B subunit in the assembly of the NMDA receptor

N-methyl-D-aspartate (NMDA) receptors play crucial roles in excitatory synaptic transmission as well as in excitotoxicity. A growing body of evidence suggests that the regulation of both subunit composition and the number of NMDA receptors reaching the surface membrane are tightly regulated. Recently, we have shown that the third membrane domains (M3) of both NR1 and NR2B subunits contain endoplasmic reticulum (ER) retention signals that prevent the unassembled subunits from leaving the ER. Furthermore, these membrane domains together with NR1 M4 are necessary for negating the ER retention signals found in M3 of NR1 and NR2B. In this addendum, we present new electrophysiological data showing that mutation of the HLFY motif, located immediately after M4 of the NR2B subunit, abolishes the surface trafficking of full-length NR1/NR2B complexes (supporting previous immunofluorescent experiments from our lab); however, the deletion of the NR2B C-terminus including the HLFY motif did not affect the formation of functional receptors when two pieces of the NR2B subunit, NR2B truncated before M4 and NR2B M4, were co-expressed together with the NR1 subunit. These observations will help to uncover the processes involved in the assembly of NR1 and NR2 subunits into functional NMDA receptors.     

Masking of Transmembrane‐Based Retention Signals Controls ER Export of γ‐Secretase

γ‐Secretase is critically involved in the Notch pathway and in Alzheimer's disease. The four subunits of γ‐secretase assemble in the endoplasmic reticulum (ER) and unassembled subunits are retained/retrieved to the ER by specific signals. We here describe a novel ER‐retention/retrieval signal in the transmembrane domain (TMD) 4 of presenilin 1, a subunit of γ‐secretase. TMD4 also is essential for complex formation, conferring a dual role for this domain. Likewise, TMD1 of Pen2 is bifunctional as well. It carries an ER‐retention/retrieval signal and is important for complex assembly by binding to TMD4. The two TMDs directly interact with each other and mask their respective ER‐retention/retrieval signals, allowing surface transport of reporter proteins. Our data suggest a model how assembly of Pen2 into the nascent γ‐secretase complex could mask TMD‐based ER‐retention/retrieval signals to allow plasma membrane transport of fully assembled γ‐secretase.     

The localization of the ER retrieval sequence for the calcium pump SERCA1

A number of studies using chimeric constructs made by fusing endoplasmic/sarcoplasmic reticulum calcium pump (SERCA) sequences with those of the plasma membrane located calcium pump (PMCA) have suggested that the retention/retrieval signal responsible for maintaining SERCA in the endoplasmic reticulum (ER) is located within the N-terminus of these pumps. Because of the difficulties in identifying the presence of constructs at the plasma membrane we have used a trans-Golgi network (TGN) marker to evaluate whether chimeric proteins are retained by the ER or have lost their retention/retrieval sequences and are able to enter the wider endomembrane system and reach the TGN. In this study, attempts to locate this retention/retrieval sequence demonstrate that the retention sequences are located not in the N-terminus, as previously suggested, but in the largely transmembranous C-terminal domain of SERCA. Further attempts to identify the precise retention/retrieval motif using SERCA1/PMCA3 chimeras were unsuccessful. This may be due to the fact that introducing SERCA1 sequences into the C-terminal PMCA3 sequence and vice versa disrupts the organization of the closely packed transmembrane helices leading to retention of such constructs by the quality control mechanisms of the ER. An alternative explanation is that SERCAs have targeting motifs that are non-linear, being made up of several segments of sequence to form a patch that interacts with the retrieval machinery.     

Assembly of nicotinic alpha7 subunits in Xenopus oocytes is partially blocked at the tetramer level

The assembly of nicotinic alpha1beta1gammadelta, alpha3beta4, and alpha7 receptors and 5-hydroxytryptamine 3A (5HT3A) receptors was comparatively evaluated in Xenopus oocytes by blue native PAGE analysis. While alpha1betagammadelta subunits, alpha3beta4 subunits, and 5HT3A subunits combined efficiently to pentamers, alpha7 subunits existed in various assembly states including trimers, tetramers, pentamers, and aggregates. Only alpha7 subunits that completed the assembly process to homopentamers acquired complex-type carbohydrates and appeared at the cell surface. We conclude that Xenopus oocytes have a limited capacity to guide the assembly of alpha7 subunits, but not 5HT3A subunits to homopentamers. Accordingly, ER retention of imperfectly assembled alpha7 subunits rather than inefficient routing of fully assembled alpha7 receptors to the cell surface limits surface expression levels of alpha7 nicotinic acetylcholine receptors.     

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.