Characterization of the two heavy chains of the T3 complex on the surface of human T lymphocytes

The T3 complex has been defined by a group of monoclonal antibodies which react with all human peripheral blood T lymphocytes and a subpopulation of thymocytes. This membrane structure includes glycoproteins of 44 (alpha), 37 (beta), 25 (gamma), and 20 kDa (delta) as well as a nonglycosylated polypeptide of 20 kDa (epsilon). The characterization of the alpha and beta chains has been of particular interest because they may constitute the T cell receptor for antigen. Here we show that the T3 complex prepared by immunoprecipitation from T lymphocytes of a leukemic patient (Sezary syndrome) displays an unusually strong association of the alpha and beta chains with the 20/25-kDa T3 proteins. The alpha and beta chains (48 and 44 kDa) were co-precipitated by anti-20-kDa T3 monoclonal antibodies as a disulfide-linked 90-kDa heterodimer. A minor 220-kDa multimer composed of proteins similar to the alpha and beta chains was also present in these immunoprecipitates. This multimer could be independently precipitated with a new monoclonal antibody WT-31, which detects the larger polypeptide chains of the T3 complex on all human T lymphocytes. After removal of N-linked oligosaccharides, both the alpha and beta chain were found to have 33-kDa peptide backbones with distinct isoelectric points. Using a monoclonal reagent T40/25, a 90-kDa heterodimer, consisting of 40- and 49-kDa chains with peptide backbones of 34 kDa was found to be T3-associated on the T leukemic cell line HPB-ALL. When the alpha and beta chains from the Sezary patient were compared with the corresponding chains from HPB-ALL by peptide mapping, large differences were observed. Taken together, the data presented here provide strong evidence that the T cell receptor for antigen is part of the T3 complex on the surface of human T lymphocytes.     

Characterization of heterotrimeric G protein complexes in rice plasma membrane

Two genes in the rice genome were identified as those encoding the gamma subunits, gamma1 and gamma2, of heterotrimeric G proteins. Using antibodies against the recombinant proteins for the alpha, beta, gamma1, and gamma2 subunits of the G protein complexes, all of the subunits were proven to be localized in the plasma membrane in rice. Gel filtration of solubilized plasma membrane proteins showed that all of the alpha subunits were present in large protein complexes (about 400 kDa) containing the other subunits, beta, gamma1, and gamma2, and probably also some other proteins, whereas large amounts of the beta and gamma (gamma1 and gamma2) subunits were freed from the large complexes and took a 60-kDa form. A yeast two-hybrid assay and co-immunoprecipitation experiments showed that the beta subunit interacted tightly with the gamma1 and gamma2 subunits, and so the beta and gamma subunits appeared to form dimers in rice cells. Some dimers were associated with the alpha subunit, because few beta, gamma1, and gamma2 subunits were present in the 400-kDa complexes in a rice mutant, d1, which was lacking in the alpha subunit. When a constitutively active form of the alpha subunit was prepared by the exchange of one amino acid residue and introduced into d1, the mutagenized subunit was localized in the plasma membrane of the transformants and took a free, and not the 400-kDa, form.     

Chemical cross-linking of bovine retinal transducin and cGMP phosphodiesterase

The bifunctional reagents para-phenyldimaleimide and maleimidobenzoyl-N-hydroxysuccinimide ester were used to chemically cross-link the subunits of the transducin and cGMP phosphodiesterase (PDE) complexes of bovine rod photoreceptor cells. The cross-linked products were identified by Western immunoblotting using antisera against purified subunits of transducin (T alpha and T beta gamma) and PDE. Oligomeric cross-linked products of transducin subunits as large as (T alpha beta gamma)3 were observed in the latent form of transducin with bound GDP. In addition to the expected T alpha beta and T beta gamma cross-linked products, a (T alpha gamma)2 structure was detected. The close proximity of T alpha and T gamma suggests that T gamma may play a role in conferring the specificity of the interaction between T alpha and rhodopsin. Most of the oligomeric cross-linked structures between T alpha and T beta gamma were diminished in the activated form of transducin, with guanosine 5'-(beta, gamma-imidotriphosphate) (Gpp(NH)p) bound. However, cross-linking between T beta and T gamma was not altered. These results suggest that transducin exists as an oligomer in solution which dissociates upon the binding of Gpp(NH)p. To identify the possible interacting domains between the T alpha, T beta, and T gamma subunits, the cross-linked products were subjected to limited tryptic proteolysis. Several cross-linked tryptic peptides of transducin subunits were found and include the cross-linked products of the N terminus 15-kDa fragment of T beta and the C terminus 5-kDa fragment of T alpha, T gamma and the 12-kDa fragment of T alpha, T gamma and the 15-kDa as well as the 23-kDa fragments of T beta, and an intra-T alpha cross-linked product of the 2- and 21-kDa fragments. These results have allowed the construction of a topographical model for the transducin subunits. The organization of the subunits of PDE (P alpha, P beta, and P gamma) was also studied. The formation of the high molecular size cross-linked products of PDE resulted in the concurrent loss of the P beta and P gamma subunits, suggesting that they are in close proximity. Finally, the interaction between transducin and PDE was examined by chemical cross-linking of transducin-Gpp(NH)p and PDE. Two additional cross-linked products of 180 and 210 kDa were obtained which could be due to the cross-linking of T alpha or T beta with P alpha beta subunits.(ABSTRACT TRUNCATED AT 250 WORDS)     

The heterotetrameric architecture of the epithelial sodium channel (ENaC).

The epithelial sodium channel (ENaC) is a key element for the maintenance of sodium balance and the regulation of blood pressure. Three homologous ENaC subunits (alpha, beta and gamma) assemble to form a highly Na+-selective channel. However, the subunit stoichiometry of ENaC has not yet been solved. Quantitative analysis of cell surface expression of ENaC alpha, beta and gamma subunits shows that they assemble according to a fixed stoichiometry, with alpha ENaC as the most abundant subunit. Functional assays based on differential sensitivities to channel blockers elicited by mutations tagging each alpha, beta and gamma subunit are consistent with a four subunit stoichiometry composed of two alpha, one beta and one gamma. Expression of concatameric cDNA constructs made of different combinations of ENaC subunits confirmed the four subunit channel stoichiometry and showed that the arrangement of the subunits around the channel pore consists of two alpha subunits separated by beta and gamma subunits.     

Phase separation of the receptor for immunoglobulin E and its subunits in Triton X-114

Above its critical micelle concentration, Triton X-114 in solution forms two phases at room temperature: a lower phase containing supramicellar aggregates and an upper phase largely depleted of detergent. This property of the detergent is potentially useful for separating under mild conditions proteins that bind detergent from those that do not (Bordier, C. (1981) J. Biol. Chem. 256, 1604-1607). We studied the distribution of the receptor for immunoglobulin E (IgE) and its subunits in the two phases. IgE and IgE complexed either with intact receptors or with the alpha chains of the receptor alone are principally partitioned into the upper phase, whereas the unliganded receptor as well as the isolated alpha, and especially the beta and gamma chains of the receptor, preferentially partition into the lower detergent phase. Chromatography of IgE and of the subunits of the receptor on a hydrophobic support showed that the beta and gamma chains have a considerably greater hydrophobic surface than the alpha chains or IgE. These results indicate that the distribution of a protein in the two phases of phase-separated Triton X-114 is not an all-or-none effect based upon whether it binds detergent or not. Rather, it reflects the overall balance between the hydrophobic and hydrophilic properties of the protein's surface.     

Subunit arrangement of gamma-aminobutyric acid type A receptors

The GABA(A) receptors are ligand-gated chloride channels. The subunit stoichiometry of the receptors is controversial; four, five, or six subunits per receptor molecule have been proposed for alphabeta receptors, whereas alphabetagamma receptors are assumed to be pentamers. In this study, alpha-beta and beta-alpha tandem cDNAs from the alpha1 and beta2 subunits of the GABA(A) receptor were constructed. We determined the minimal length of the linker that is required between the two subunits for functional channel expression for each of the tandem constructs. 10- and 23-amino acid residues are required for alpha-beta and beta-alpha, respectively. The tandem constructs either alone or in combination with each other failed to express functional channels in Xenopus oocytes. Therefore, we can exclude tetrameric or hexameric alphabeta GABA(A) receptors. We can also exclude proteolysis of the tandem constructs. In addition, the tandem constructs were combined with single alpha, beta, or gamma subunits to allow formation of pentameric arrangements. In contrast to the combination with alpha subunits, the combination with either beta or gamma subunits led to expression of functional channels. Therefore, a pentameric arrangement containing two alpha1 and three beta2 subunits is proposed for the receptor composed of alpha and beta subunits. Our findings also favor an arrangement betaalphagammabetaalpha for the receptor composed of alpha, beta, and gamma subunits.     

Major pertussis-toxin-sensitive GTP-binding protein of bovine lung. Purification, characterization and production of specific antibodies

Two GTP-binding proteins which can be ADP-ribosylated by islet-activating protein, pertussis toxin, were purified from the cholate extract of bovine lung membranes. Both proteins had the same heterotrimeric structure (alpha beta gamma), but the alpha subunits were dissociated from the beta gamma when they were purified in the presence of AlCl3, MgCl2 and NaF. The molecular mass of the alpha subunit of the major protein (designated GLu, with beta gamma) was 40 kDa and that of the minor one was 41 kDa. The results of peptide mapping analysis of alpha subunits with a limited proteolysis indicated that GLu alpha was entirely different from the alpha of brain Gi or Go, while the 41-kDa polypeptide was identical with the alpha of bovine brain Gi. The kinetics of guanosine 5'-[3-O-thio]triphosphate (GTP[gamma S]) binding to GLu was similar to that to lung Gi but quite different from that to brain Go. On the other hand, incubation of GLu alpha at 30 degrees C caused a rapid decrease of GTP[gamma S] binding, the inactivation curve being similar to that of Go alpha but different from that of Gi alpha. The alpha subunits of lung Gi and GLu did not react with the antibodies against the alpha subunit of bovine brain Go. The antibodies were raised in rabbits against GLu alpha and were purified with a GLu alpha-Sepharose column. The purified antibodies reacted not only with GLu alpha but also with the 41-kDa protein and purified brain Gi alpha. However, the antibodies adsorbed with brain Gi alpha reacted only with GLu alpha, indicating antisera raised with GLu alpha contained antibodies that recognize both Gi alpha and GLu alpha, and those specific to GLu alpha. These results further indicate that GLu is different from Gi or Go. Anti-GLu alpha antibodies reacted with the 40-kDa proteins in the membranes of bovine brain and human leukemic (HL-60) cells. The beta gamma subunits were also purified from bovine lung. The beta subunit was the doublet of 36-kDa and 35-kDa polypeptides. The lung beta gamma could elicit the ADP-ribosylation of GLu alpha by islet-activating protein, increase the GTP[gamma S] binding to GLu and protect the thermal denaturation of GLu alpha. The antibodies raised against brain beta gamma cross-reacted with lung beta but not with lung gamma.     

Characterization of truncated alpha chain products from human, rat, and mouse high affinity receptor for immunoglobulin E

The high affinity receptor for immunoglobulin E (IgE) is a tetrameric structure (alpha beta gamma 2) consisting of non-covalently associated subunits: one IgE-binding alpha chain, one 4-fold membrane spanning beta chain, and two disulfide-linked gamma chains. Here, we have engineered alpha cDNA constructs (alpha trunc) encoding exclusively the leader peptide and the extracellular domain of the alpha subunit. Transfection of human alpha trunc into COS-7 cells resulted in the secretion of soluble IgE-binding polypeptides. By contrast, the polypeptides generated from rat and mouse alpha trunc transfections were sequestered in the endoplasmic reticulum and degraded even though they appeared to fold properly as judged by their capacity to bind IgE. Stable transfectants with human alpha trunc were obtained from a dihydrofolate reductase-deficient Chinese hamster ovary cell line. Several clones secreted substantial amounts (0.1 microgram/ml/10(6) cells) of IgE-binding polypeptides. The dissociation rate of bound IgE from this soluble truncated alpha (kappa-1 = 4.9 x 10(-6) s-1 at 25 degrees C) was characteristic of receptors on intact cells. After treatment with tunicamycin, the transfectants secreted unglycosylated 18-kDa polypeptides which could also bind IgE. These unglycosylated products had a tendency to form dimers and higher oligomers which were resistant to treatment by sodium dodecyl sulfate and reducing agents. These data demonstrate unequivocally that the extracellular domain of the alpha subunit is sufficient to mediate high affinity binding of IgE. Furthermore, posttranslational addition of carbohydrates is not required for proper folding and function of the receptor binding site. The truncated human alpha should be a suitable reagent for crystallographic analysis and for detailed analysis of the receptor binding sites.     

The gamma and epsilon subunits of the CD3 complex inhibit pre-Golgi degradation of newly synthesized T cell antigen receptors

The T cell receptor for antigen (TCR) is composed of six different transmembrane proteins. T cells carefully control the intracellular transport of the receptor and allow only complete receptors to reach the plasma membrane. In an attempt to understand how T cells regulate this process, we used c-DNA transfection and subunit-specific antibodies to follow the intracellular transport of five subunits (alpha beta gamma delta epsilon) of the receptor. In particular, we assessed the intracellular stability of each chain. Our results showed that the chains were markedly different in their susceptibility to intracellular degradation. TCR alpha and beta and CD3 delta were degraded rapidly, whereas CD3 gamma and epsilon were stable. An analysis of the N-linked oligosaccharides of the glycoprotein subunits suggested that the chains were unable to reach the medial Golgi during the metabolic chase. This was supported by immunofluorescence micrographs that showed both the stable CD3 gamma and unstable CD3 delta chain localized in the endoplasmic reticulum. To study the effects of subunit associations on intracellular transport we used cotransfection to reconstitute precise combinations of subunits. Associations between stable and unstable subunits expressed in the same cell led to the formation of stable complexes. These complexes were retained in or close to the endoplasmic reticulum. The results suggested that the intracellular transport of the T cell receptor could be regulated by two mechanisms. The TCR alpha and beta and CD3 delta subunits were degraded rapidly and as a consequence failed to reach the plasma membrane. CD3 gamma or epsilon were stable but were retained inside the cell. The results also demonstrated that there was an interplay between the two pathways such that the CD3 gamma and epsilon subunits were able to protect labile chains from rapid intracellular degradation. In this way, they could seed subunit assembly in or close to the endoplasmic reticulum and allow a stable receptor to form before its transport to the plasma membrane.     

Stoichiometry of a ligand-gated ion channel determined by fluorescence energy transfer

We have developed a method to determine the stoichiometry of subunits within an oligomeric cell surface receptor using fluorescently tagged antibodies to the individual subunits and measuring energy transfer between them. Anti-c-Myc monoclonal antibody (mAb 9-E10) derivatized with a fluorophore (europium cryptate, EuK) was used to individually label c-Myc-tagged alpha1-, beta2-, or gamma2-subunits of the hetero-oligomeric gamma-aminobutyric acid (GABAA) receptor in intact cells. The maximal fluorescent signal derived from the alpha1(c-Myc)beta2gamma2 and the alpha1beta2(c-Myc)gamma2 receptors was twice that obtained with alpha1beta2gamma2(c-Myc), suggesting that there are 2x alpha-, 2x beta-, and 1x gamma-subunits in a receptor monomer. This observation was extended using fluorescence energy transfer. Receptors were half-maximally saturated with EuK-anti-c-Myc mAb, and the remaining alpha1(c-Myc) subunits were labeled with excess anti-c-Myc mAb derivatized with the fluorescence energy acceptor, XL665. On exposure to laser light, energy transfer from EuK to XL665 occurred with alpha1(c-Myc)beta2gamma2 and alpha1beta2(c-Myc)gamma2, but no significant energy transfer was observed with alpha1beta2gamma2(c-Myc) receptors, indicating the absence of a second gamma-subunit in a receptor monomer. We confirm that the GABAA receptor subtype, alpha1beta2gamma2, is composed of two copies each of the alpha- and beta-subunits and one copy of the gamma-subunit (i.e. (alpha1)2(beta2)2(gamma2)1) and conclude that this method would have general applicability to other multisubunit cell surface proteins.     

Identification of exposed and buried determinants of the membrane-bound acetylcholine receptor from Torpedo californica

The ability of five rabbit anti-acetylcholine receptor antisera to recognize the membrane-bound receptor from Torpedo californica has been investigated. Two antisera, raised against affinity-purified native receptor, react extensively with purified receptor-rich membrane vesicles. Since the membrane vesicles are impermeable to macromolecules and are oriented right side out, these two antisera recognize predominantly extracellular determinants. Two antisera against sodium dodecyl sulfate denatured receptor and one against purified delta subunit react poorly with the membrane-bound receptor. Only 10-20% of the determinants recognized by these antisera are accessible to antibodies when the receptor is membrane bound. Many of the latent sites can be exposed by permeabilizing the vesicles with saponin, by alkaline extraction of the membranes to remove peripheral proteins, or by a combination of these two treatments. These treatments neither solubilize the receptors nor interfere with their ability to undergo agonist-induced affinity changes. Subunit analysis of the sites on the membrane-bound receptor that are accessible to antibodies indicates that the alpha, beta, and delta chains possess extracellular determinants. Buried sites are present on all four of the subunits. Saponin permeabilization makes latent sites accessible on alpha and delta while alkaline extraction uncovers determinants on alpha, gamma, and delta. Treatment of membranes by both procedures reveals sites on beta, gamma, and delta that are not uncovered by either treatment alone. This study, in conjunction with results from other laboratories demonstrating that the gamma chain is extracellularly exposed, suggests that all four subunits are transmembrane proteins.     

Interaction between GABAA receptor subunit intracellular loops: implications for higher order complex formation

The majority of fast inhibitory neurotransmission in the CNS is mediated by the GABA type-A (GABAA) receptor, a ligand-gated chloride channel. Of the approximately 20 different subunits composing the hetero-pentameric GABAA receptor, the gamma2 subunit in particular seems to be important in several aspects of GABAA receptor function, including clustering of the receptor at synapses. In this study, we report that the intracellular loop of the gamma2 subunit interacts with itself as well as with gamma1, gamma3 and beta1-3 subunits, but not with the alpha subunits. We further show that gamma2 subunits interact with photolabeled pentameric GABAA receptors composed of alpha1, beta2/3 and gamma2 subunits, and calculate the dissociation constant to be in the micromolar range. By using deletion constructs of the gamma2 subunit in a yeast two-hybrid assay, we identified a 23-amino acid motif that mediates self-association, residues 389-411. We confirmed this interaction motif by inhibiting the interaction in a glutathione-S-transferase pull-down assay by adding a corresponding gamma2-derived peptide. Using similar approaches, we identified the interaction motif in the gamma2 subunit mediating interaction with the beta2 subunit as a 47-amino acid motif that includes the gamma2 self-interacting motif. The identified gamma2 self-association motif is identical to the interaction motif reported between GABAA receptor and GABAA receptor-associated protein (GABARAP). We propose a model for GABAA receptor clustering based on GABARAP and GABAA receptor subunit-subunit interaction.     

Characterization of acetylcholine receptor subunits in developing and in denervated mammalian muscle

We have used subunit-specific antibodies to identify and to characterize partially the alpha, beta, gamma, and delta subunits of rat skeletal muscle acetylcholine receptor (AChR) on immunoblots. The alpha subunit of rat muscle is a single band of 42 kDa, whereas the beta subunit has an apparent molecular mass of 48 kDa. Both alpha and beta subunits are glycosylated and contain one or more N-linked oligosaccharide chains that are sensitive to endoglycosidase H digestion. The gamma and delta subunits, on the other hand, each appear as doublets on immunoblots, with apparent molecular masses of 52 kDa (gamma), 48 kDa (gamma') and 58 kDa (delta), 53 kDa (delta'), respectively. In each case, the two bands are structurally related and the lower band is probably the partial degradation product of the corresponding upper band. Each of the four gamma and delta polypeptides is N-glycosylated and contains both endoglycosidase H-sensitive and endoglycosidase H-resistant oligosaccharides. When the AChRs purified from embryonic, neonatal, adult, and denervated adult rat muscles were compared, no differences in the mobilities of alpha, beta, or delta subunits on sodium dodecyl sulfate gels were detected among them, either with or without endoglycosidase treatment. The gamma subunits, which were present in AChRs purified from neonatal, embryonic, or denervated rat muscles, were also identical; no gamma subunit was detected, however, in AChRs of normal adult rat muscle.     

The third subunit of desulfoviridin-type dissimilatory sulfite reductases.

In addition to the 50-kDa (alpha) and 40-kDa (beta) subunits, an 11-kDa polypeptide has been discovered in highly purified Desulfovibrio vulgaris (Hildenborough) dissimilatory sulfite reductase. This is in contrast with the hitherto generally accepted alpha 2 beta 2 tetrameric subunit composition. Purification, high-ionic-strength gel-filtration, native electrophoresis and isoelectric focussing do not result in dissociation of the 11-kDa polypeptide from the complex. Densitometric scanning of SDS gels and denaturing gel-filtration indicate a stoichiometric occurrence. A similar 11-kDa polypeptide is present in the desulfoviridin of D. vulgaris oxamicus (Monticello), D. gigas and D. desulfuricans ATCC 27774. We attribute an alpha 2 beta 2 gamma 2 subunit structure to desulfoviridin-type sulfite reductases. N-terminal sequences of the alpha, beta and gamma subunits are reported.     

Structural relationships among class I isozymes of human liver alcohol dehydrogenase

The alpha subunit of human liver alcohol dehydrogenase has been submitted to structural analysis. Together with earlier work on the beta and gamma subunits, the results allow conclusions on the relationship of all known forms of the class I type of the enzyme. Two segments of the alpha subunit were determined; one was also reinvestigated in the beta and gamma subunits. The results establish 11 residue replacements among class I subunits in the segments analyzed and show that the alpha, beta, and gamma protein chains each are structurally distinct in the active site regions, where replacements affect positions influencing coenzyme binding (position 47; Gly in alpha, Arg in beta and gamma) and substrate specificity (position 48; Thr in alpha and beta, Ser in gamma). Residue 128, previously not detected in beta and gamma subunits, corresponds to a position of another isozyme difference (Arg in beta and gamma, Ser in alpha). The many amino acid replacements in alcohol dehydrogenases even at their active sites illustrate that in judgements of enzyme functions absolute importance of single residues should not be overemphasized. Available data suggest that alpha and gamma are the more dissimilar forms within the family of the three class I subunits that have resulted from two gene duplications. The class distinction of alcohol dehydrogenases previously suggested from enzymatic, electrophoretic, and immunological properties therefore also holds true in relation to their structures.     

Preferential assembly of epithelial sodium channel (ENaC) subunits in Xenopus oocytes: role of furin-mediated endogenous proteolysis

The epithelial sodium channel (ENaC) is preferentially assembled into heteromeric alphabetagamma complexes. The alpha and gamma (not beta) subunits undergo proteolytic cleavage by endogenous furin-like activity correlating with increased ENaC function. We identified full-length subunits and their fragments at the cell surface, as well as in the intracellular pool, for all homo- and heteromeric combinations (alpha, beta, gamma, alphabeta, alphagamma, betagamma, and alphabetagamma). We assayed corresponding channel function as amiloride-sensitive sodium transport (I(Na)). We varied furin-mediated proteolysis by mutating the P1 site in alpha and/or gamma subunit furin consensus cleavage sites (alpha(mut) and gamma(mut)). Our findings were as follows. (i) The beta subunit alone is not transported to the cell surface nor cleaved upon assembly with the alpha and/or gamma subunits. (ii) The alpha subunit alone (or in combination with beta and/or gamma) is efficiently transported to the cell surface; a surface-expressed 65-kDa alpha ENaC fragment is undetected in alpha(mut)betagamma, and I(Na) is decreased by 60%. (iii) The gamma subunit alone does not appear at the cell surface; gamma co-expressed with alpha reaches the surface but is not detectably cleaved; and gamma in alphabetagamma complexes appears mainly as a 76-kDa species in the surface pool. Although basal I(Na) of alphabetagamma(mut) was similar to alphabetagamma, gamma(mut) was not detectably cleaved at the cell surface. Thus, furin-mediated cleavage is not essential for participation of alpha and gamma in alphabetagamma heteromers. Basal I(Na) is reduced by preventing furin-mediated cleavage of the alpha, but not gamma, subunits. Residual current in the absence of furin-mediated proteolysis may be due to non-furin endogenous proteases.     

Surface expression of mutated subunits of the high affinity mast cell receptor for IgE

The mast cell receptor with high affinity for IgE consists of four transmembrane polypeptides which are held together by detergent-sensitive interactions: an IgE-binding alpha chain, a single beta chain, and a disulfide-linked dimer of gamma chains. Now that the cDNAs that code for each of the subunits have been isolated, it should be possible to probe by site-specific mutations, which portions of the receptor are critical for transmembrane signaling. One prerequisite for such studies is that the mutant receptors be expressible on the cell surface. We have explored this issue by transiently transfecting COS 7 cells with mutant subunits and assessing surface expression by IgE binding. Removal of any single cytoplasmic domain of the receptor's subunits had little influence on surface expression, and even receptors missing all five cytoplasmic domains were expressed, albeit less efficiently. Minor changes within the transmembrane domains (TMs) sometimes produced major effects and more drastic changes in the TMs ablated surface expression entirely. These data suggest that the TMs are critical loci for receptor display. Cys7 (residue 2 in the gamma TM) was shown to form the inter-gamma disulfide bond and to be nonessential for surface expression. By localizing this bond, residues in the TM of gamma that are buried in the interface between the gamma subunits could be predicted. Consistent with observations on other membrane proteins (Rees, D. C., DeAntonio, L., and Eisenberg, D. (1989) Science 245, 510-513), maximal interspecies conservation was observed for those residues in the gamma TM predicted to be buried. This was also true for those residues in the alpha and beta TMs predicted to be buried by analysis of the TM hydrophobic moments.     

The molecular structure of different polymorphic forms of canine C3 and C4

The subunit composition of different electrophoretic forms of canine C3 and C4 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of reduced immune precipitates. Canine C4 comprises alpha, beta, and gamma chains of approximate molecular weight 90 000–95 000, 72 000, and 33 000, respectively. The molecular weight of the alpha chain of the C4 1 allelic product was approximately 95 000, but 90 000 for the C4 2 and C4 4 allotypes. No differences were observed in the molecular weights of the beta or gamma chains of any canine C4 phenotype tested. Canine C3 appears to be encoded by a single locus. The subunit composition comprises an alpha and beta chain with molecular weights of approximately 106 000 and 71000, respectively. Unlike C4, no differences in the molecular weights of the subunits were observed in different electrophoretic forms of canine C3.     

A novel site on gamma 3 subunits important for assembly of GABA(A) receptors

Gamma-aminobutyric acid, type A (GABA(A)) receptors are ligand-gated chloride channels and are the major inhibitory transmitter receptors in the central nervous system. The majority of these receptors is composed of two alpha, two beta, and one gamma subunits. To identify sequences important for subunit assembly, we generated C-terminally truncated and chimeric gamma(3) constructs. From their ability to associate with full-length alpha(1) and beta(3) subunits, we concluded that amino acid sequence gamma(3)(70-84) either directly interacts with alpha(1) or beta(3) subunits or stabilizes a contact site elsewhere in the protein. The observation that this sequence contains amino acid residues homologous to gamma(2) residues contributing to the benzodiazepine-binding site at the alpha(1)/gamma(2) interface suggested that in alpha(1)beta(3)gamma(3) receptors the sequence gamma(3)(70-84) is located at the alpha(1)/gamma(3) interface. In the absence of alpha(1) subunits this sequence might allow assembly of beta(3) with gamma(3) subunits. Other experiments indicated that sequences gamma(3)(86-95) and gamma(3)(94-107), which are homologous to previously identified sequences important for assembly of gamma(2) subunits, are also important for assembly of gamma(3) subunits. This indicates that during assembly of the GABA(A) receptor, more than one N-terminal sequence is important for binding to the same neighboring subunit. Whether the three sequences investigated are involved in direct interaction or stabilize other regions involved in intersubunit contacts has to be further studied.