The reconstituted alpha 3 beta 3 delta complex of the thermostable F1-ATPase

Previously we reported that ATPase activity was recovered when the subunit alpha + beta + gamma or alpha + beta + delta of the F1-ATPase from the thermophilic bacterium PS3 were combined under appropriate conditions. Unlike that of holoenzyme (TF1) and the alpha + beta + gamma mixture, ATPase activity of the alpha + beta + delta mixture was heat labile and insensitive to azide inhibition (Yoshida, M., Sone, N., Hirata, H., and Kagawa, Y. (1977) J. Biol. Chem. 252, 3480-3485). Here, the properties of purified subunit complexes were compared in detail with those of native TF1. The subunit stoichiometries of the complexes were determined to be alpha 3 beta 3 gamma 1 and alpha 3 beta 3 delta 1. In general, the properties of the alpha 3 beta 3 gamma complex are very similar to those of TF1, whereas those of the alpha 3 beta 3 delta complex are significantly different. ATPase activity of the alpha 3 beta 3 delta complex is cold labile. The alpha 3 beta 3 delta complex showed a less stringent specificity for substrate and divalent cation than TF1 and the alpha 3 beta 3 gamma complex. Two Km values for ATP were exhibited by the alpha 3 beta 3 delta complex with the lower one being in the range of 0.1 microM. Equilibrium dialysis experiments revealed that the alpha 3 beta 3 delta complex cannot specifically bind ADP in the absence of Mg2+, while TF1 and the alpha 3 beta 3 gamma complex bind about 1 and 3 mol of ADP/mol of enzyme, respectively. ADP-dependent inactivation of the alpha 3 beta 3 delta complex by dicyclohexylcarbodiimide was not observed. The alpha 3 beta 3 gamma complex was readily formed when the gamma subunit was added to the alpha 3 beta 3 delta complex, suggesting that the alpha 3 beta 3 delta complex is not a "dead-end" complex. The cause of thermolability of the alpha 3 beta 3 delta complex appears to be the low stability of the complex itself at high temperature and not due to an unusually low thermostability of the delta subunit.     

Blue native PAGE as a useful method for the analysis of the assembly of distinct combinations of nicotinic acetylcholine receptor subunits

Oligomerization of complete and incomplete combinations of rat muscle-type nicotinic acetylcholine receptor (nAChR) subunits in Xenopus oocytes was studied by blue native PAGE and compared with acetylcholine-activated current in these cells. The rank order of expression level judged by current was alpha 1 beta 1 gamma delta > alpha 1 beta 1 gamma > alpha 1 beta 1 delta > alpha 1 gamma delta > alpha 1 delta > alpha 1 gamma. alpha 1 and alpha 1 beta 1 were not functional. Protein complexes incorporating a heptahistidyl-tagged alpha 1 subunit were chromatographically purified from digitonin extracts of oocytes and resolved by blue native PAGE. In the absence of any co-expressed nAChR subunit, the majority of alpha 1 formed aggregates. Co-expression of beta 1 had no effect on alpha 1 aggregation, whereas both gamma and delta diminished alpha 1 aggregation in favor of discrete oligomers: alpha 1 formed tetramers together with gamma and dimers, trimers, and tetramers together with delta. When alpha 1 gamma was complemented with beta 1 to form a functional alpha 1 beta 1 gamma receptor, a small amount of a pentamer was found besides a prominent alpha 1-His7 beta 1 gamma trimer. Expression of the functional alpha 1 beta 1 delta receptor yielded marked amounts of a pentamer besides dimers and trimers. These results are discussed in terms of the assembly model of Green and Claudio (Cell 74, 57-69, 1994), substantiating that blue native PAGE is suited for the investigation of ion channel assembly.     

Adoptively transferred gamma delta T cells indirectly regulate murine graft-versus-host reactivity following donor leukocyte infusion therapy in mice

The purpose of this study was to determine whether gamma delta T cells were able to regulate graft-vs-host (GVH) reactivity mediated by alpha beta T cells in murine recipients transplanted with MHC-mismatched marrow grafts. Studies were conducted using ex vivo-activated gamma delta T cells because this was a more clinically relevant strategy, and these cells have been shown to be capable of facilitating alloengraftment without causing GVH disease (GVHD). Coadministration of activated gamma delta T cells and naive alpha beta T cells at the time of bone marrow transplantation (BMT) significantly exacerbated GVHD when compared with naive alpha beta T cells alone. In contrast, when the administration of naive alpha beta T cells was delayed for 2 wk post-BMT, survival was significantly enhanced in mice transplanted with BM plus activated gamma delta T cells vs those given marrow cells alone. Mitigation of GVHD by activated gamma delta T cells occurred only at high doses (150 x 106) and was a unique property of gamma delta T cells, as activated alpha beta T cells were incapable of ameliorating the subsequent development of GVHD. Protection from GVHD was not due to the direct inhibition of naive alpha beta T cells by gamma delta T cells. Rather, gamma delta T cells mediated this effect indirectly through donor BM-derived alpha beta T cells that acted as the proximate regulatory population responsible for the decrease in GVH reactivity. Collectively, these data demonstrate that activated gamma delta T cells are capable of modulating the ability of MHC-incompatible nontolerant alpha beta T cells to cause GVHD after allogeneic BMT.     

Acetylcholine receptor assembly is stimulated by phosphorylation of its gamma subunit

Different combinations of Torpedo acetylcholine receptor (AChR) subunits stably expressed in mouse fibroblasts were used to establish a role for phosphorylation in AChR biogenesis. When cell lines expressing fully functional AChR complexes (alpha 2 beta gamma delta) were labeled with 32P, only gamma and delta subunits were phosphorylated. Forskolin, which causes a 2- to 3-fold increase in AChR expression by stimulating subunit assembly, increased unassembled gamma phosphorylation, but had little effect on unassembled delta. The forskolin effect on subunit phosphorylation was rapid, significantly preceding its effect on expression. The pivotal role of the gamma subunit was established by treating alpha beta gamma and alpha beta delta cell lines with forskolin and observing increased expression of only alpha beta gamma complexes. This effect was also observed in alpha gamma, but not alpha delta cells. We conclude that the cAMP-induced increase in expression of cell surface AChRs is due to phosphorylation of unassembled gamma subunits, which leads to increased efficiency of assembly of all four subunits.     

Reconstitution of adenosine triphosphatase of thermophilic bacterium from purified individual subunits.

1. Five subunits (alpha, beta, gamma, delta, and epsilon) of an ATPase from a thermophilic bacterium PS3 were purified in the presence of 8 M urea by ion exchange chromatography. Then the ATPase activity was reconstituted by mixing the subunit solutions and incubating them at 20-45 degrees, at pH 6.3 to 7.0. 2. Mixtures containing beta + gamma or alpha + beta + delta regained ATP-hydrolyzing activity, but mixtures of alpha + beta and beta + delta did not. Combinations not including beta were all inactive. 3. The ATPase activity reconstituted from alpha + beta + delta was thermolabile and insensitive to NaN3, whereas the activities obtained from mixtures containing beta and gamma were thermostable and sensitive to NaN3, like the native ATPase. 4. The assemblies containing both beta and gamma subunits had the same mobility as the native ATPase molecule on gel electrophoresis, those without the gamma subunit moved more rapidly toward the anode. 5. Subunits epsilon and delta did not inhibit the ATPase activity of either the assembly (alpha + beta + gamma) or the native ATPase.     

Assembly of Torpedo acetylcholine receptors in Xenopus oocytes

To study pathways by which acetylcholine receptor (AChR) subunits might assemble, Torpedo alpha subunits were expressed in Xenopus oocytes alone or in combination with beta, gamma, or delta subunits. The maturation of the conformation of the main immunogenic region (MIR) on alpha subunits was measured by binding of mAbs and the maturation of the conformation of the AChR binding site on alpha subunits was measured by binding of alpha-bungarotoxin (alpha Bgt) and cholinergic ligands. The size of subunits and subunit complexes was assayed by sedimentation on sucrose gradients. It is generally accepted that native AChRs have the subunit composition alpha 2 beta gamma delta. Torpedo alpha subunits expressed alone resulted in an amorphous range of complexes with little affinity for alpha Bgt or mAbs to the MIR, rather than in a unique 5S monomeric assembly intermediate species. A previously recognized temperature-dependent failure in alpha subunit maturation may cause instability of the monomeric assembly intermediate and accumulation of aggregated denatured alpha subunits. Coexpression of alpha with beta subunits also resulted in an amorphous range of complexes. However, coexpression of alpha subunits with gamma or delta subunits resulted in the efficient formation of 6.5S alpha gamma or alpha delta complexes with high affinity for mAbs to the MIR, alpha Bgt, and small cholinergic ligands. These alpha gamma and alpha delta subunit pairs may represent normal assembly intermediates in which Torpedo alpha is stabilized and matured in conformation. Coexpression of alpha, gamma, and delta efficiently formed 8.8S complexes, whereas complexes containing alpha beta and gamma or alpha beta and delta subunits are formed less efficiently. Assembly of beta subunits with complexes containing alpha gamma and delta subunits may normally be a rate-limiting step in assembly of AChRs.     

Substrate and inhibitor activities of the screw sense isomers of metal-nucleotide complexes in the formyltetrahydrofolate synthetase reaction

Phosphorothioate analogues of ATP and isomers of CrATP and CrADP were used to examine the nucleotide stereoselectivity of formyltetrahydrofolate synthetase from procaryotic and eucaryotic sources. Substrate activity of the thio-ATP analogues increased as the site of sulfur substitution was changed from the gamma to the alpha position. Thus, adenine nucleotide analogues substituted with sulfur at an alpha nonbridging position (ATP alpha S isomers) were the most active, and ATP gamma S was inactive. When Mg2+ was used as the divalent cation, both enzymes showed a clear preference (higher V/Km value) for the Sp isomer of ATP beta S although the magnitude of the preference was greater with the bacterial enzyme. With Cd2+ as the divalent cation the Rp isomer was preferred, but the difference was greater with the yeast enzyme. Both (Sp)-MgATP beta S and (Rp)-CdATP beta S have the delta or right-hand screw sense configuration of the metal chelate ring. The reversal of stereoselectivity when the cation was changed indicates that the metal ion is coordinated to the beta-phosphate group. No stereoselectivity was observed when ATP alpha S isomers were used in the presence of Mg2+ or Cd2+, suggesting that the metals are not coordinated to the alpha-phosphate. ATP beta S was also found to be a competitive inhibitor of MgATP and CdATP, and the lowest Ki values were obtained with the lambda screw sense isomers. The screw sense isomers of bidentate CrATP exhibited no detectable substrate activity but were competitive inhibitors of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gamma delta T cells assist alpha beta T cells in adoptive transfer of contact sensitivity.

Cutaneous immune responses to contact sensitizers such as picryl chloride or oxazolone, are classical manifestations of T cell-mediated immunity in vivo. In fact, the first documentation of T cell-mediated immunity was the ability to adoptively transfer contact sensitivity (CS) responses. Although it is now clear that Ag/MHC-restricted alpha beta TCR positive effector T cells are responsible for 24 to 48 h CS responses, other subsets of Thy-1+ cells in mice also participate in the elicitation of CS. Thus, Thy-1+, CD5+, CD3-, B220+, hapten-specific, non-MHC-restricted early-acting cells are required to initiate CS responses by leading to local serotonin release, which allows for extravascular recruitment of the late-acting, alpha beta TCR+, CS effector T cells. This study describes another T cell population that is needed for the adoptive transfer of CS by alpha beta T cells. In vitro treatment of a mixture of CS effector cells with hamster mAb to gamma delta TCR, together with rabbit complement, or by panning on anti-hamster Ig-coated dishes, diminished substantially the subsequent transfer of CS reactivity without affecting either CS-initiating cells, or the later-acting, alpha beta TCR+ CS effector T cells. Immune cells treated with anti-alpha beta TCR mAb, or recovered as adherent cells from petri dishes after anti-gamma delta TCR panning (i.e., gamma delta TCR-enriched cells), reconstituted the ability of anti-gamma delta TCR-treated immune cells (i.e., alpha beta TCR-enriched cells) to transfer 24-h CS responsiveness. The phenotype of the gamma delta T cells that assisted CS effector alpha beta T cells was: CD3+, CD4-, and CD8+. The gamma delta T cells that assisted alpha beta T cells were not Ag-specific since anti-alpha beta-TCR-treated cells (gamma delta T-enriched) from picryl chloride immunized donors aided alpha beta T cells (anti-gamma delta TCR-treated) from oxazolone-immunized donors, and conversely gamma delta T cells from oxazolone-immunized donors aided alpha beta T cells from picryl chloride immunized donors. Furthermore, the CS-regulating gamma delta T cells were not MHC-restricted because gamma delta T cells from H2d or H2b donors could assist alpha beta T cells from H2k donors. It was concluded that a regulatory population of non-Ag specific, non-MHC-restricted gamma delta T cells was needed to assist immune effector, Ag/MHC-specific alpha beta T cells in the adoptive transfer of CS.     

Heterogeneity of the retinal G-protein transducin from frog rod photoreceptors. Biochemical identification and characterization of new subunits.

Transducin, a retinal G-protein, has been shown to exist as heterotrimers of alpha (39,000), beta (36,000), and gamma (approximately 7,000) subunits. Blue Sepharose CL-6B column chromatography of a transducin preparation extracted with a metal-free, low salt buffer containing GTP showed three distinct alpha and two distinct beta gamma activities in frog (Rana catesbeiana) rod outer segment. The binding of a hydrolysis-resistant GTP analog in these alpha fractions was proportional to the amount of the M(r) 39,000 protein. The first alpha was eluted in a complex with an inhibitory subunit of cGMP phosphodiesterase, but alpha subunits in the second and the third fractions were not complexed with any proteins. Two-dimensional gel electrophoresis and characterization with regard to the interaction with the inhibitory subunit of cGMP phosphodiesterase suggested that the first and the second alpha s were the same protein; however, the third alpha showed different characters as follows. We designated alpha in the first two fractions as alpha 1, and alpha in the third fraction as alpha 2. Nonlinear regression analysis for the binding of a hydrolysis-resistant GTP analog to both alpha subunits revealed a single class of GTP binding sites with an apparent stoichiometry of 1 mol of GTP/mol of alpha. Compared with alpha 1, alpha 2 required larger amounts of rhodopsin and beta gamma for the binding of a hydrolysis-resistant GTP analog. alpha 2 also showed less binding with the inhibitory subunit of cGMP phosphodiesterase. Both alpha 1 and alpha 2 complexed with beta gamma or beta delta (described below) were substrates for pertussis toxin-dependent ADP-ribosylation. The protein profiles of two beta gamma fractions revealed that the main fraction was composed of a beta gamma complex; however, the second active fraction was composed of beta complexed with delta (M(r) 12,000). Compared with beta gamma, beta delta stimulated GTP binding to alpha 1 at approximately 10-fold higher concentration. Two-dimensional gel electrophoresis revealed five beta and two gamma isoforms in beta gamma. Only one beta isoform was present in beta delta. The diversity of transducin subunits may reflect different signaling pathways in visual signal transduction.     

Impaired platelet aggregation and sustained bleeding in mice lacking the fibrinogen motif bound by integrin alpha IIb beta 3.

Blood loss at sites of vascular rupture is controlled by the adhesion and aggregation of platelets and the formation of an insoluble fibrin matrix. Fibrinogen is considered to be critical in these processes by both providing an abundant dimeric ligand for alpha IIb beta 3-mediated platelet aggregation, and serving as the fundamental building block of the fibrin polymer. To establish an in vivo model system to examine in detail the importance of alpha IIb beta 3-fibrinogen interactions in platelet function, hemostasis, response to injury and vasoocclusive disease, and to test the prevailing hypothesis that the C-terminal segment of the fibrinogen gamma chain is essential for alpha IIb beta 3 binding, we have used gene-targeting technology in mice to eliminate the last five residues (QAGDV) from the gamma chain. Mice homozygous for the modified gamma chain gene (gamma delta 5/gamma delta 5) displayed a generally normal hematological profile, including normal platelet count, plasma fibrinogen level, clotting time and fibrin crosslinking. However, both gamma delta 5-fibrinogen binding to alpha IIb beta 3 and platelet aggregation were highly defective. Remarkably, another alpha IIb beta 3-dependent process, clot retraction, was unaffected by the gamma delta 5 mutation. Despite the preservation of clotting function, gamma delta 5/gamma delta 5 mice were unable to control blood loss following a surgical challenge and occasionally developed fatal neonatal bleeding events.     

T cell receptor gamma gene status of human alpha/beta+ and gamma/delta+ T cell clones: absence of V9JP rearrangements in alpha/beta+ clones is not a result of a lack of rearrangements involving more 5' J gamma segments

T cell receptor (TCR) gamma gene rearrangements were examined in panels of human T cell clones expressing TCR alpha/beta or gamma/delta heterodimers. Over half of the alpha/beta+ clones had both chromosomes rearranged to C gamma 2 but this was the case for only 20% of the gamma/delta+ clones. While more than half of the gamma/delta+ clones showed a V9JP rearrangement, this configuration was absent from all 49 alpha/beta+ clones analysed. However, this was not a result of all rearrangements being to the more 3' J gamma genes as 11 alpha/beta+ clones had rearrangement(s) to JP1, the most 5' J gamma gene segment. Both alpha/beta+ and gamma/delta+ clones showed a similar pattern of V gamma gene usage in rearrangements to J gamma 1 or J gamma 2 with a lower proportion of the more 3' genes being rearranged to J gamma 2 than for the more 5' genes. Several alpha/beta+ and several gamma/delta+ clones had noncoordinate patterns of rearrangement involving both C gamma 1 and C gamma 2. Eleven out of fourteen CD8+ clones tested had both chromosomes rearranged to C gamma 2 whereas all clones derived from CD4-8- cells and having unconventional phenotypes (CD4-8- or CD4+8+) had at least one C gamma 1 rearrangement. Twelve out of twenty-seven CD4+ clones also had this pattern, suggesting that CD4-8+ clones had a tendency to utilize more 3' J gamma gene segments than CD4+ clones. There was some evidence for interdonor variation in the proportions of TCR gamma rearrangements to C gamma 1 or C gamma 2 in alpha/beta+ clones as well as gamma/delta+ clones. The results illustrate the unique nature of the V9JP rearrangement in gamma/delta+ clones and the possible use of a sequential mechanism of TCR gamma gene rearrangements during T cell differentiation is discussed.     

alpha4beta3delta GABA(A) receptors characterized by fluorescence resonance energy transfer-derived measurements of membrane potential

Selective modulators of gamma-aminobutyric acid, type A (GABA(A)) receptors containing alpha(4) subunits may provide new treatments for epilepsy and premenstrual syndrome. Using mouse L(-tk) cells, we stably expressed the native GABA(A) receptor subunit combinations alpha(3)beta(3)gamma(2,) alpha(4)beta(3)gamma(2), and, for the first time, alpha(4)beta(3)delta and characterized their properties using a novel fluorescence resonance energy transfer assay of GABA-evoked depolarizations. GABA evoked concentration-dependent decreases in fluorescence resonance energy transfer that were blocked by GABA(A) receptor antagonists and, for alpha(3)beta(3)gamma(2) and alpha(4)beta(3)gamma(2) receptors, modulated by benzodiazepines with the expected subtype specificity. When combined with alpha(4) and beta(3), delta subunits, compared with gamma(2), conferred greater sensitivity to the agonists GABA, 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP), and muscimol and greater maximal efficacy to THIP. alpha(4)beta(3)delta responses were markedly modulated by steroids and anesthetics. Alphaxalone, pentobarbital, and pregnanolone were all 3-7-fold more efficacious at alpha(4)beta(3)delta compared with alpha(4)beta(3)gamma(2.) The fluorescence technique used in this study has proven valuable for extensive characterization of a novel GABA(A) receptor. For GABA(A) receptors containing alpha(4) subunits, our experiments reveal that inclusion of delta instead of gamma(2) subunits can increase the affinity and in some cases the efficacy of agonists and can increase the efficacy of allosteric modulators. Pregnanolone was a particularly efficacious modulator of alpha(4)beta(3)delta receptors, consistent with a central role for this subunit combination in premenstrual syndrome.     

Alpha beta lineage-specific expression of the alpha T cell receptor gene by nearby silencers

T cells expressing either the alpha beta or gamma delta antigen receptor (TCR) are distinct cell lineages. The single locus encoding the TCR alpha and delta genes requires special regulation to avoid alpha gene expression in gamma delta T cells. We show here that the minimal alpha enhancer is active in the gamma delta T cell lineage but gains alpha beta lineage specificity through negative cis-acting elements 3' of the C alpha gene that silence the enhancer in gamma delta T cells. The negative elements at the C alpha locus consist of several silencers that work in an orientation- and distance-independent fashion. These silencers also act on a retroviral enhancer that is normally ubiquitously expressed, restricting its activity to alpha beta cells. The alpha silencers are active in non-T cell lines, suggesting that the decision of a cell to differentiate into the alpha beta T cell lineage may involve specific relief from these silencers. Silencers are likely to be as important as enhancers in establishing lineage-specific gene expression in many systems.     

Expression of an Inwardly Rectifying Potassium Channel in Xenopus Oocytes

Localization of the gamma and delta types of mRNAs for Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) was determined in the rat brain, making use of in situ hybridization histochemistry. The gamma and delta mRNAs as well as the alpha and beta mRNAs for CaM-kinase II were heterogeneously and distinctly distributed. In the Purkinje cell layer of the cerebellum, alpha, beta, and gamma mRNAs but not delta mRNA were present, whereas beta, gamma, and delta mRNAs were present in the locus coeruleus. These findings provide evidence that CaM-kinase II exists in a variety of forms in different cells composed of a variable number and type of subunits.     

A delta T-cell receptor deleting element transgenic reporter construct is rearranged in alpha beta but not gamma delta T-cell lineages.

T cells can be divided into two groups on the basis of the expression of either alpha beta or gamma delta T-cell receptors (TCRs). Because the TCR delta chain locus lies within the larger TCR alpha chain locus, control of the utilization of these two receptors is important in T-cell development, specifically for determination of T-cell type: rearrangement of the alpha locus results in deletion of the delta coding segments and commitment to the alpha beta lineage. In the developing thymus, a relative site-specific recombination occurs by which the TCR delta chain gene segments are deleted. This deletion removes all D delta, J delta, and C delta genes and occurs on both alleles. This delta deletional mechanism is evolutionarily conserved between mice and humans. Transgenic mice which contain the human delta deleting elements and as much internal TCR delta chain coding sequence as possible without allowing the formation of a complete delta chain gene were developed. Several transgenic lines showing recombinations between deleting elements within the transgene were developed. These lines demonstrate that utilization of the delta deleting elements occurs in alpha beta T cells of the spleen and thymus. These recombinations are rare in the gamma delta population, indicating that the machinery for utilization of delta deleting elements is functional in alpha beta T cells but absent in gamma delta T cells. Furthermore, a discrete population of early thymocytes containing delta deleting element recombinations but not V alpha-to-J alpha rearrangements has been identified. These data are consistent with a model in which delta deletion contributes to the implementation of a signal by which the TCR alpha chain locus is rearranged and expressed and thus becomes an alpha beta T cell.     

Neurotoxins Distinguish Between Different Neuronal Nicotinic Acetylcholine Receptor Subunit Combinations

Neuronal and muscle nicotinic acetylcholine receptor subunit combinations expressed in Xenopus oocytes were tested for sensitivity to various neurotoxins. Extensive blockade of the alpha 3 beta 2 neuronal subunit combination was achieved by 10 nM neuronal bungarotoxin. Partial blockade of the alpha 4 beta 2 neuronal and alpha 1 beta 1 gamma delta muscle subunit combinations was caused by 1,000 nM neuronal bungarotoxin. The alpha 2 beta 2 neuronal subunit combination was insensitive to 1,000 nM neuronal bungarotoxin. Nearly complete blockade of all neuronal subunit combinations resulted from incubation with 2 nM neosurugatoxin, whereas 200 nM neosurugatoxin was required for partial blockade of the alpha 1 beta 1 gamma delta muscle subunit combination. The alpha 2 beta 2 and alpha 3 beta 2 neuronal subunit combinations were partially blocked by 10,000 nM lophotoxin analog-1, whereas complete blockade of the alpha 4 beta 2 neuronal and alpha 1 beta 1 gamma delta muscle subunit combinations resulted from incubation with this concentration of lophotoxin analog-1. The alpha 1 beta 1 gamma delta muscle subunit combination was blocked by the alpha-conotoxins G1A and M1 at concentrations of 100 nM. All of the neuronal subunit combinations were insensitive to 10,000 nM of both alpha-conotoxins. Thus, neosurugatoxin and the alpha-conotoxins distinguish between muscle and neuronal subunit combinations, whereas neuronal bungarotoxin and lophotoxin analog-1 distinguish between different neuronal subunit combinations on the basis of differing alpha subunits.     

gamma delta T cells in the human intestine express surface markers of activation and are preferentially located in the epithelium

We have investigated the expression of the alpha beta and the gamma delta T cell receptor (TCR) in the human intestine. By immunohistology we found that 39% of CD3+ intraepithelial lymphocytes (IEL) expressed the gamma delta TCR compared to 3% of CD3+ lamina propria lymphocytes (LPL). Cytofluorometric analysis of isolated cells revealed a significantly higher proportion of gamma delta T cells among CD3+ IEL compared to LPL and peripheral blood lymphocytes. This was due to an increase in both CD8+ (low density) and CD4-CD8- gamma delta T cells in IEL. Most alpha beta IEL expressed high-density CD8. About 30% of both IEL and LPL expressed CD25 (alpha-chain of the IL-2 receptor). HML-1 expression was detected on nearly all IEL and on 27% of LPL. CD25 and HML-1 were preferentially expressed on intestinal alpha beta and gamma delta T cells, respectively. Thus, human gamma delta T cells are located preferentially in the gut epithelium and are phenotypically different from alpha beta T cells, which constitute the majority of both LPL and IEL.     

Assembly of the mammalian muscle acetylcholine receptor in transfected COS cells

We have investigated the mechanisms of assembly and transport to the cell surface of the mouse muscle nicotinic acetylcholine receptor (AChR) in transiently transfected COS cells. In cells transfected with all four subunit cDNAs, AChR was expressed on the surface with properties resembling those seen in mouse muscle cells (Gu, Y., A. F. Franco, Jr., P.D. Gardner, J. B. Lansman, J. R. Forsayeth, and Z. W. Hall. 1990. Neuron. 5:147-157). When incomplete combinations of AChR subunits were expressed, surface binding of 125I-alpha-bungarotoxin was not detected except in the case of alpha beta gamma which expressed less than 15% of that seen with all four subunits. Immunoprecipitation and sucrose gradient sedimentation experiments showed that in cells expressing pairs of subunits, alpha delta and alpha gamma heterodimers were formed, but alpha beta was not. When three subunits were expressed, alpha delta beta and alpha gamma beta complexes were formed. Variation of the ratios of the four subunit cDNAs used in the transfection mixture showed that surface AChR expression was decreased by high concentrations of delta or gamma cDNAs in a mutually competitive manner. High expression of delta or gamma subunits also each inhibited formation of a heterodimer with alpha and the other subunit. These results are consistent with a defined pathway for AChR assembly in which alpha delta and alpha gamma heterodimers are formed first, followed by association with the beta subunit and with each other to form the complete AChR.     

The unfolding story of T cell receptor gamma

Antigen-specific, major histocompatibility complex-restricted recognition by classical T cells is mediated by a T cell receptor (TCR) consisting of a disulfide-linked alpha beta heterodimer. During the search for the genes encoding the alpha and beta proteins, a third immunoglobulin-like gene, termed gamma, was uncovered. Like the TCR alpha and beta genes, the TCR gamma gene consists of variable and constant segments that rearrange during T cell development in the thymus. Although the physiological role of TCR gamma remains an enigma, much has been learned with the recent identification of the protein products of this gene family in both mice and humans. The gamma chain is associated with a partner chain, termed delta. The gamma delta heterodimer is associated with an invariant T3 complex, very similar to that associated with the alpha beta heterodimer, and appears predominantly, if not exclusively, on cells with a CD4-, CD8- phenotype both in the thymus and in the periphery. TCR gamma delta is the first T3-associated receptor to appear during thymocyte development and defines a separate T cell lineage distinct from alpha beta-bearing cells. Although TCR alpha beta-bearing cells and TCR gamma delta-bearing cells follow parallel developmental pathways, the diversity of expressed gamma delta receptors is extremely limited relative to that of alpha beta receptors.