Cloning and sequencing of the equine and ovine high-affinity IgE receptor β-and γ-chain cDNA

The high-affinity receptor for IgE is expressed on the surface of mast cells and basophils. It is a transmembrane protein with one alpha, one beta and two gamma subunits. The cDNA sequences for the alpha subunit have already been determined. We report here the cDNA sequences for the beta and gamma subunits. The cytoplasmic domains of these subunits are important for intracellular signalling and the deduced amino acid sequences show the expected immunoreceptor tyrosine-based activation motifs. The gamma subunit is highly conserved between species but more variation is seen with the beta subunit. Near the C terminus of the equine beta chain there is a two-base deletion, which changes the reading frame: residue 237 (human numbering) becomes Asp instead of Glu and the chain is three amino acids shorter than the other known mammalian and rodent sequences.     

Role of the transmembrane and cytoplasmic domains in the assembly and surface exposure of the platelet integrin GPIIb/IIIa.

Integrins are alpha beta heterodimers that play a major role in cell-cell contacts and in interactions between cells and extracellular matrices. Identification of structural domains that are critical for the expression of such receptors at the cell surface in a functional conformation is one of the major issues that has not yet been resolved. In the present study, the role of the cytoplasmic and transmembrane domains of each of the subunits has been examined using platelet GPIIb/IIIa as a prototypic integrin. GPIIb/IIIa (alpha IIb/beta 3) is a member of the integrin family and functions as a receptor for fibrinogen, fibronectin, von Willebrand factor, and vitronectin at the surface of activated platelets. Human megakaryocyte GPIIb and GPIIIa cDNAs were used to create a GPIIb mutant coding for the extracellular GPIIb heavy chain alone (GPIIb delta 1) and a GPIIIa mutant lacking the transmembrane and cytoplasmic domains (GPIIIa delta m). Full length and mutant cDNAs were subcloned into the expression vector pECE and used to transfect COS cells. The formation of heterodimers and their cellular localization was analyzed by immunoprecipitation and immunofluorescence labeling using anti-platelet GPIIb/IIIa antibodies. We show here that the extracellular domains of alpha and beta subunits are able to form a heterodimer, although with a lower efficiency, in the absence of the transmembrane and cytoplasmic domains. The presence of the cytoplasmic and transmembrane domains in the alpha subunit is, however, necessary for expression at the surface of the cell whereas the corresponding domains of the beta subunit are not required.     

Lysine 271 in the transmembrane domain of the T-cell antigen receptor beta chain is necessary for its assembly with the CD3 complex but not for alpha/beta dimerization

The T-cell antigen receptor (TcR) complex present on most T-cells is formed by a clone-specific disulfide-linked alpha/beta heterodimer noncovalently associated to the CD3 complex, the latter composed of five invariant polypeptides: gamma, delta, epsilon, zeta/zeta, or zeta/eta. The presence of conserved, oppositely charged, amino acids in the predicted transmembrane domains of all the subunits of the TcR.CD3 complex suggests that these residues may have a critical function in the assembly and/or stabilization of the complex. In order to analyze the role of the transmembrane-charged amino acids in the association and cell surface expression of the TcR.CD3 complex, we have carried out site-directed mutagenesis of Lys271 in the transmembrane domain of the TcR beta chain and analyzed the capacity of the altered chain to assemble in a TcR beta-negative T-cell line. Here we show that substitution of this positively charged residue by alanine or glutamine does not prevent cytoplasmic association of alpha and beta chains to form disulfide-linked heterodimers, but does abolish formation of an alpha/beta.CD3 complex and, consequently, its expression on the cell surface.     

Role of conserved prolines in the structure and function of the Na+/dicarboxylate cotransporter 1, NaDC1

The Na+/dicarboxylate cotransporter 1 (NaDC1) is a low-affinity transporter for citric acid cycle intermediates such as succinate and citrate. The sequence of NaDC1 contains a number of conserved proline residues in predicted transmembrane helices (TMs) 7 and 10. These transmembrane domains are of particular importance because they may be involved in determining the substrate or cation-binding affinity in NaDC1. Four conserved proline residues in TMs 7 and 10 of rabbit NaDC1 were replaced with alanine to promote ideal alpha helix or glycine to promote free conformation, and the mutant transporters were expressed in the HRPE cell line. Mutations of prolines in TM 10 produced decreased protein expression and activity, whereas mutations of prolines in TM 7 completely abolished protein expression and activity. The chemical chaperone glycerol was found to improve the expression of the Pro-351 mutants in TM 7, suggesting that these mutants had defects in trafficking. The inactive mutant transporters at position 351 could also be rescued by the addition of a proline at a second site. For example, the P351A-F347P mutant had restored activity, although its substrate specificity was altered. We conclude that, in TM 7, Pro-327 may be of particular importance in the function of the transporter, whereas Pro-351 may affect protein targeting. The prolines in TM 10, at positions 523 and 524, may not be directly involved in the transporter function but may be necessary for maintaining structure.     

Complete structure of the mouse mast cell receptor for IgE (Fc epsilon RI) and surface expression of chimeric receptors (rat-mouse-human) on transfected cells

The high affinity receptor for IgE (Fc epsilon RI) found on mast cells and basophils is a tetrameric complex of a single alpha subunit, a single beta subunit, and two identical gamma subunits. The genes for the three subunits of mouse Fc epsilon RI have now been cloned from the mast cell line, PT18. When compared at the DNA level, the rat and mouse subunits are similarly conserved. However, at the protein level the homology between mouse and rat alpha is surprisingly low (71% identities) especially in the cytoplasmic regions (57% identities) which are of different length (25 and 20 residues, respectively). By contrast the beta and gamma are homogeneously conserved between mouse and rat (83 and 93% identities, respectively). The consensus amino acid sequence of the alpha subunit derived from three species (rat, mouse, and human) shows that the cytoplasmic tail diverges to the same extent as the leader peptide. Conversely, the transmembrane domain of the alpha is highly conserved and contains 10 consecutive residues that are identical. Comparisons between mouse Fc epsilon RI and other mouse proteins reveal regions of high homology between the alpha subunit and Fc gamma RIIa and between the gamma subunit and the zeta chain of the T cell receptor. Cells transfected with the alpha gene express the alpha subunit on their surface very inefficiently. Efficient expression is only achieved after co-transfection of the three rodent genes or of the human alpha gene together with the rodent gamma without apparent need for beta. The subunits are completely interchangeable upon transfection so that various chimeric mouse-rat-human receptors can be expressed.     

Identification of amino acid residues important for assembly of GABA receptor alpha1 and gamma2 subunits

Comparative models of GABA(A) receptors composed of alpha1 beta3 gamma2 subunits were generated using the acetylcholine-binding protein (AChBP) as a template and were used for predicting putative engineered cross-link sites between the alpha1 and the gamma2 subunit. The respective amino acid residues were substituted by cysteines and disulfide bond formation between subunits was investigated on co-transfection into human embryonic kidney (HEK) cells. Although disulfide bond formation between subunits could not be observed, results indicated that mutations studied influenced assembly of GABA(A) receptors. Whereas residue alpha1A108 was important for the formation of assembly intermediates with beta3 and gamma2 subunits consistent with its proposed location at the alpha1(+) side of GABA(A) receptors, residues gamma2T125 and gamma2P127 were important for assembly with beta3 subunits. Mutation of each of these residues also caused an impaired expression of receptors at the cell surface. In contrast, mutated residues alpha1F99C, alpha1S106C or gamma2T126C only impaired the formation of receptors at the cell surface when co-expressed with subunits in which their predicted interaction partner was also mutated. These data are consistent with the prediction that the mutated residue pairs are located close to each other.     

A cDNA presumptively coding for the alpha subunit of the receptor with high affinity for immunoglobulin E

Rat mast cells and a neoplastic analogue such as rat basophilic leukemia (RBL) cells have receptors that have exceptionally high affinity for immunoglobulin E (IgE). When aggregated, these receptors induce cellular degranulation. The alpha chain of the receptor contains the binding site for IgE; the function(s) of the noncovalently associated beta and gamma chains is (are) still undefined. Using a cDNA library constructed from the mRNA of RBL cells, we have isolated a cDNA clone whose sequence predicts a putative 23-residue signal peptide, followed by a sequence that accurately predicts the amino acid composition, the peptide molecular weight, and six peptide sequences (encompassing 59 residues or 26% of the total number) determined for the alpha chain by direct analysis. These findings provide strong evidence that the cDNA codes for the alpha chain, even though expression has not been unambiguously achieved. The sequence suggests that the alpha chain contains a 180-residue extracellular portion with two homologous domains of approximately 35 residues, a 20-residue transmembrane segment containing an aspartic acid, and a 27-residue cytoplasmic portion containing 9 basic amino acids. The sequence shows no homology with the low-affinity receptor for IgE from lymphocytes but over 30% homology with an Fc gamma receptor.     

The alpha subunit of the human IgE receptor (FcERI) is sufficient for high affinity IgE binding

The alpha subunit of the FcERI binds IgE with high affinity. Previous studies have demonstrated that alpha subunit expression requires the presence of beta and/or gamma subunits, and it is not known how these two subunits contribute to the ability of the alpha subunit to bind IgE. In this report, we describe the expression and characterization of a human chimeric alpha subunit. The data demonstrate that high affinity IgE binding does not require the presence of the beta and/or gamma subunits and that this activity is localized to the extracellular domain (residues 26-201) of the human alpha subunit. Permanent cell lines expressing the chimeric receptor were used to characterize the binding parameters of the alpha subunit. These cell lines provide a means of identifying therapeutic agents which may be effective in the treatment/management of allergic diseases.     

Role of CD3 gamma in T cell receptor assembly

The T cell receptor (TCR) consists of the Ti alpha beta heterodimer and the associated CD3 gamma delta epsilon and zeta 2 chains. The structural relationships between the subunits of the TCR complex are still not fully known. In this study we examined the role of the extracellular (EC), transmembrane (TM), and cytoplasmic (CY) domain of CD3 gamma in assembly and cell surface expression of the complete TCR in human T cells. A computer model indicated that the EC domain of CD3 gamma folds as an Ig domain. Based on this model and on alignment studies, two potential interaction sites were predicted in the EC domain of CD3 gamma. Site-directed mutagenesis demonstrated that these sites play a crucial role in TCR assembly probably by binding to CD3 epsilon. Mutagenesis of N-linked glycosylation sites showed that glycosylation of CD3 gamma is not required for TCR assembly and expression. In contrast, treatment of T cells with tunicamycin suggested that N-linked glycosylation of CD3 delta is required for TCR assembly. Site-directed mutagenesis of the acidic amino acid in the TM domain of CD3 gamma demonstrated that this residue is involved in TCR assembly probably by binding to Ti beta. Deletion of the entire CY domain of CD3 gamma did not prevent assembly and expression of the TCR. In conclusion, this study demonstrated that specific TCR interaction sites exist in both the EC and TM domain of CD3 gamma. Furthermore, the study indicated that, in contrast to CD3 gamma, glycosylation of CD3 delta is required for TCR assembly and expression.     

Identification and functional characterization of the zeta-chain dimerization motif for TCR surface expression.

We recognized a common dimerization motif between the transmembrane (TM) domain of zeta-chain family members and glycophorin A. We have shown that a glycine within the zeta-dimerization motif is critical for zeta-homodimerization and also for its association with the TCR/CD3 complex. Similarly, two residues within the CD3 delta gamma TM domains have proven to be critical for their interaction with the zeta-homodimer. A three-dimensional homology model of the zeta-chain TM domain highlights potential residues preferentially involved either in the zeta 2-CD3 or zeta 2-TCR alpha beta association, confirming our experimental findings. These results indicate that, for symmetrical reasons, the zeta-homodimer participates in the TCR/CD3 complex assembly by interacting with CD3 gamma delta TM domains, thereby masking their degradation signals located in the cytoplasmic tails.     

Different transmembrane domains associate with distinct endoplasmic reticulum components during membrane integration of a polytopic protein

We have been studying the insertion of the seven transmembrane domain (TM) protein opsin to gain insights into how the multiple TMs of polytopic proteins are integrated at the endoplasmic reticulum (ER). We find that the ER components associated with the first and second TMs of the nascent opsin polypeptide chain are clearly distinct. The first TM (TM1) is adjacent to the alpha and beta subunits of the Sec61 complex, and a novel component, a protein associated with the ER translocon of 10 kDa (PAT-10). The most striking characteristic of PAT-10 is that it remains adjacent to TM1 throughout the biogenesis and membrane integration of the full-length opsin polypeptide. TM2 is also found to be adjacent to Sec61alpha and Sec61beta during its membrane integration. However, TM2 does not form any adducts with PAT-10; rather, a transient association with the TRAM protein is observed. We show that the association of PAT-10 with opsin TM1 does not require the N-glycosylation of the nascent chain and occurs irrespective of the amino acid sequence and transmembrane topology of TM1. We conclude that the precise makeup of the ER membrane insertion site can be distinct for the different transmembrane domains of a polytopic protein. We find that the environment of a particular TM can be influenced by both the "stage" of nascent chain biosynthesis reached, and the TM's relative location within the polypeptide.     

Arg-274 and Leu-277 of the gamma-aminobutyric acid type A receptor alpha 2 subunit define agonist efficacy and potency

Alanine-scanning mutagenesis and the whole cell voltage clamp technique were used to investigate the function of the extracellular loop between the second and third transmembrane domains (TM2-TM3) of the gamma-aminobutyric acid type A receptor (GABA(A)-R). A conserved arginine residue in the TM2-TM3 loop of the GABA(A)-R alpha(2) subunit was mutated to alanine, and the mutant alpha(2)(R274A) was co-expressed with wild-type beta(1) and gamma(2S) subunits in human embryonic kidney (HEK) 293 cells. The GABA EC(50) was increased by about 27-fold in the mutant receptor relative to receptors containing a wild-type alpha(2) subunit. Similarly, the GABA EC(50) at alpha(2)(L277A)beta(1)gamma(2S) and alpha(2)(K279A)beta(1)gamma(2S) GABA(A)-R combinations was increased by 51- and 4-fold, respectively. The alpha(2)(R274A) or alpha(2)(L277A) mutations also reduced the maximal response of piperidine-4-sulfonic acid relative to GABA by converting piperidine-4-sulfonic acid into a weak partial agonist at the GABA(A)-R. Based on these results, we propose that alpha(2)(Arg-274) and alpha(2)(Leu-277) are crucial to the efficient transduction of agonist binding into channel gating at the GABA(A)-R.     

Associations between subunit ectodomains promote T cell antigen receptor assembly and protect against degradation in the ER

The T cell antigen receptor (TCR) is an oligomeric protein complex made from at least six different integral membrane proteins (alpha beta gamma delta epsilon and zeta). The TCR is assembled in the ER of T cells, and correct assembly is required for transport to the cell surface. Single subunits and partial receptor complexes are retained in the ER where TCR alpha, beta, and CD3 delta chains are degraded selectively. The information required for the ER degradation of the TCR beta chain is confined to the membrane anchor of the protein (Wileman et al., 1990c; Bonifacino et al., 1990b). In this study we show that the rapid degradation of the TCR beta chain is inhibited when it assembles with single CD3 gamma, delta, or epsilon subunits in the ER, and have started to define the role played by transmembrane anchors, and receptor ectodomains, in the masking proteolytic targeting information. Acidic residues within the membrane spanning domains of CD3 subunits were essential for binding to the TCR beta chain. TCR beta chains and CD3 subunits therefore interact via transmembrane domains. However, when sites of binding were restricted to the membrane anchor of the TCR beta chain, stabilization by CD3 subunits was markedly reduced. Interactions between membrane spanning domains were not, therefore, sufficient for the protection of the beta chain from ER proteolysis. The presence of the C beta domain, containing the first 150 amino acids of the TCR ectodomain, greatly increased the stability of complexes formed in the ER. For assembly with CD3 epsilon, stability was further enhanced by the V beta amino acids. The results showed that the efficient neutralization of transmembrane proteolytic targeting information required associations between membrane spanning domains and the presence of receptor ectodomains. Interactions between receptor ectodomains may slow the dissociation of CD3 subunits from the beta chain and prolong the masking of transmembrane targeting information. In addition, the close proximity of TCR and CD3 ectodomains within the ER may provide steric protection from the action of proteases within the ER lumen.     

Lactoperoxidase-catalyzed iodination of the receptor for immunoglobulin E at the cytoplasmic side of the plasma membrane

The structural organization of the high affinity receptor for immunoglobulin E has been investigated in plasma membrane vesicles from rat basophilic leukemia cells using lactoperoxidase-catalyzed 125I-iodination to label exposed polypeptide regions. Intact vesicles are predominantly right-side-out in orientation, and lactoperoxidase iodination of these vesicles results in labeling of the alpha subunit of receptor but not the beta and gamma subunits. Lysis of these vesicles to expose the cytoplasmic face of the membrane by two different methods permits labeling of the beta and gamma subunits with no increase in labeling of alpha. The results indicate that both the beta and gamma subunits of the receptor have segments exposed at the cytoplasmic side of the plasma membrane. These studies have also revealed a previously unidentified IgE binding component in the membrane vesicles; its 125I-labeling characteristics and some other properties are described.     

Fc receptor gamma chain residues at the interface of the cytoplasmic and transmembrane domains affect association with FcalphaRI, surface expression, and function

The assembly of multiple subunit immunoreceptors is dependent on transmembrane interactions. The Fc receptor gamma (FcR-gamma) chain is a ubiquitous immune receptor tyrosine-based activation motif-containing dimeric subunit, gamma(2), which in humans associates with both the activating members of the leukocyte receptor cluster, including the IgA receptor FcalphaRI, and the classical Fc receptors, including the IgE receptor FcepsilonRI. This study identifies a new site in the transmembrane region of FcR-gamma that affects receptor assembly and surface expression with FcalphaRI but not with FcepsilonRI. The wild type complex, FcalphaRI-gamma(2)WT, remains robustly associated in both Brij-96 and Thesit detergent conditions. However, mutation of either Tyr(25) or Cys(26) of FcR-gamma, near the interface of the transmembrane and cytoplasmic regions, resulted in impaired FcR-gamma association with FcalphaRI. This association was disrupted in the presence of the detergent Brij-96 but was preserved in milder conditions using the detergent Thesit. Ligand-mediated cross-linking of the FcalphaRI-gamma(2)Y25F mutant receptor resulted in diminished signal transduction, including an abnormal calcium response, compared with the FcalphaRI-gamma(2)WT receptor. Furthermore, the FcalphaRI-gamma(2)Y25F mutant receptor was expressed at the cell surface at approximately 10% of that of the wild type, whereas the surface expression of FcepsilonRI-gamma(2)Y25F was not significantly different from the wild type. In contrast, although the FcalphaRI-gamma(2)C26S mutant was also less stably associated, it was not reduced in surface expression or function. Thus, these TM residues of FcR-gamma are important for association with FcalphaRI and probably other activating LRC members but not with the classical FcR, FcepsilonRI.     

Cytoplasmic and transmembrane domains of integrin beta 1 and beta 3 subunits are functionally interchangeable

Integrin beta subunits combine with specific sets of alpha subunits to form functional adhesion receptors. The structure and binding properties of integrins suggest the presence of domains controlling at least three major functions: subunit association, ligand binding, and cytoskeletal interactions. To more carefully define structure/function relationships, a cDNA construct consisting of the extracellular domain of the avian beta 1 subunit and the cytoplasmic and transmembrane domains of the human beta 3 subunit was prepared and expressed in murine 3T3 cells. The resulting chimeric beta 1/3 subunit formed heterodimers with alpha subunits from the beta 1 subfamily, could not interact with alpha IIb from the beta 3 subfamily, was targeted to focal contacts, and formed functional complexes within the focal contacts. A second cDNA construct was prepared that coded for an avian beta 1 subunit without a transmembrane or cytoplasmic domain. This subunit was not found in association with an accompanying alpha subunit, nor was it found expressed on the cell surface. Instead, it accumulated in vesicles within the cytoplasm and was eventually shed from the cell. The results from studies of the behavior of these two cDNA constructs demonstrate that the transmembrane and cytoplasmic domains play no role in alpha subunit selection, that the cytoplasmic domain of beta 3 is capable of functioning in the context of alpha subunits with which it is not normally paired, and that both integrin subunits must be membrane associated for normal assembly and transport to cell surface adhesive structures.     

An asymmetric contribution to gamma-aminobutyric type A receptor function of a conserved lysine within TM2-3 of alpha1, beta2, and gamma2 subunits

Mutations that impair the expression and/or function of gamma-aminobutyric acid type A (GABAA) receptors can lead to epilepsy. The familial epilepsy gamma2(K289M) mutation affects a basic residue conserved in the TM2-3 linker of most GABAA subunits. We investigated the effect on expression and function of the Lys --> Met mutation in mouse alpha1(K278M), beta2(K274M), and gamma2(K289M) subunits. Compared with cells expressing wild-type and alpha1beta2gamma2(K289M) receptors, cells expressing alpha1(K278M)beta2gamma2 and alpha1beta2(K274M)gamma2 receptors exhibited reduced agonist-evoked current density and reduced GABA potency, with no change in single channel conductance. The low current density of alpha1beta2(K274M)gamma2 receptors coincided with reduced surface expression. By contrast the surface expression of alpha1(K278M)beta2gamma2 receptors was similar to wild-type and alpha1beta2gamma2(K289M) receptors suggesting that the alpha1(K278M) impairs function. In keeping with this interpretation GABA-activated channels mediated by alpha1(K278M)beta2gamma2 receptors had brief open times. To a lesser extent gamma2(K289M) also reduced mean open time, whereas beta2(K274M) had no effect. We used propofol as an alternative GABAA receptor agonist to test whether the functional deficits of mutant subunits were specific to GABA activation. Propofol was less potent as an activator of alpha1(K278M)beta2gamma2 receptors. By contrast, neither beta2(K274M) nor gamma2(K289M) affected the potency of propofol. The beta2(K274M) construct was unique in that it reduced the efficacy of propofol activation relative to GABA. These data suggest that the alpha1 subunit Lys-278 residue plays a pivotal role in channel gating that is not dependent on occupancy of the GABA binding site. Moreover, the conserved TM2-3 loop lysine has an asymmetric function in different GABAA subunits.     

Transmembrane domain helix packing stabilizes integrin alphaIIbbeta3 in the low affinity state

Regulated changes in the affinity of integrin adhesion receptors ("activation") play an important role in numerous biological functions including hemostasis, the immune response, and cell migration. Physiological integrin activation is the result of conformational changes in the extracellular domain initiated by the binding of cytoplasmic proteins to integrin cytoplasmic domains. The conformational changes in the extracellular domain are likely caused by disruption of intersubunit interactions between the alpha and beta transmembrane (TM) and cytoplasmic domains. Here, we reasoned that mutation of residues contributing to alpha/beta interactions that stabilize the low affinity state should lead to integrin activation. Thus, we subjected the entire intracellular domain of the beta3 integrin subunit to unbiased random mutagenesis and selected it for activated mutants. 25 unique activating mutations were identified in the TM and membrane-proximal cytoplasmic domain. In contrast, no activating mutations were identified in the more distal cytoplasmic tail, suggesting that this region is dispensable for the maintenance of the inactive state. Among the 13 novel TM domain mutations that lead to integrin activation were several informative point mutations that, in combination with computational modeling, suggested the existence of a specific TM helix-helix packing interface that maintains the low affinity state. The interactions predicted by the model were used to identify additional activating mutations in both the alpha and beta TM domains. Therefore, we propose that helical packing of the alpha and beta TM domains forms a clasp that regulates integrin activation.     

Assembly and function of integrin receptors is dependent on opposing alpha and beta cytoplasmic domains.

The membrane proximal regions of integrin alpha and beta subunits are highly conserved in evolution. In particular, all integrin alpha subunits share the KXGFFKR sequence at the beginning of their cytoplasmic domains. Previous work has shown that this domain is important in integrin receptor assembly. Using chimeric integrin alpha and beta subunits, we show that the native cytoplasmic domains of both subunits must be present for efficient assembly. Most strikingly, chimeric alpha 1 and beta 1 subunits with reciprocally swapped intracellular domains dimerize selectively into collagen IV receptors expressed at high levels on the surface. However, these receptors, which bind ligand efficiently, are deficient in a variety of post-ligand binding events, including cytoskeletal association and induction of tyrosine phosphorylation. Furthermore, deletion of the distal alpha cytoplasmic domain in the swapped heterodimers leads to ligand-independent focal contact localization, which also occurs in wild-type subunits when the distal cytoplasmic domain is deleted. These results show that proper integrin assembly requires opposed alpha and beta cytoplasmic domains, and this opposition prevents ligand-independent focal contact localization. Our working hypothesis is that these two domains may associate during receptor assembly and provide the mechanism for integrin receptor latency.