Isolation, maturational level, and functional capacity of human colon lamina propria plasma cells.

BACKGROUND AND AIMS: Large numbers of plasma cells (PC) localise in the intestinal lamina propria (LP) where they play a critical role in the defence against pathogens. This study analyses the level of maturation reached by normal human colon LPPC in comparison with that of bone marrow (BM) PC. METHODS: A technique was designed to purify LPPC by combining collagenase digestion of the mucosal layer and immunomagnetic selection of CD54(+) LP cells. It provided highly purified PC, as demonstrated by morphology, CD38(h) phenotype, and cytoplasmic IgA staining criteria. This procedure allowed comparison of in vitro functional capacities and a broad phenotypic analysis of BMPC and LPPC. RESULTS: LPPC and BMPC exhibited identical expression of differentiation markers (CD19(-/+), CD20(-), HLA-DR(low/-), VS38c(high)), survival molecules (CD95 (low/-), Bcl-2(+)), and B cell transcription factor profile, as well as similar in vitro Ig secreting kinetics (14 days) and lack of susceptibility to apoptosis by CD95 ligation. In contrast, they markedly differed in adhesion molecule expression, as LPPC showed higher levels of CD44 and CD21 and were alpha 4 beta 7(+) whereas BMPC lacked this integrin and expressed higher levels of CD49d and CD31. CONCLUSION: These data indicate that PC at effector sites of the humoral response (BM and LP) show similar high differentiation, survival, and functional features but display a distinctive pattern of adhesion molecules, probably related to their respective homing locations.

Activity of the nicotinic acetylcholine receptor alpha5 and alpha7 subunit promoters in muscle cells.

The acetylcholine receptor alpha5 and alpha7 subunits are components of different nicotinic receptor subtypes expressed in the nervous system. However, they are also present in non-neuronal tissues. We have detected alpha5 and alpha7 transcripts in mouse C2C12 muscle cells. Moreover, on differentiation of myoblasts into myotubes, the amount of alpha7 transcripts increased significantly, whereas alpha5 remained unchanged. In order to explore how the expression of these neuronal genes is regulated in muscle, we have characterized their promoter activities. Deletion and mutagenesis analysis with transfected reporter genes showed that transcriptional activity was controlled by regulatory elements also operative in neuronal-like cells. Thus, the activity of the alpha5 subunit core promoter decreased to approximately 50% on alteration of one, two, or three of the five Sp1 binding sites present in this region and was almost abolished when four or five sites were mutated simultaneously. In the case of the alpha7 subunit promoter, the upstream stimulatory factor and the early growth response gene transcription factor were involved in regulating its transcriptional activity. In addition, the alpha7 promoter was activated during the differentiation process, in a mechanism partially dependent on the mentioned factors.

Multiple roles of the conserved key residue arginine 209 in neuronal nicotinic receptors.

We have examined the role of a highly conserved arginine (R209), which flanks the M1 transmembrane segment of nAChRs, in the biogenesis and function of neuronal nAChRs. Point mutations revealed that, in alphaBgtx-sensitive neuronal alpha7 nAChRs, the conserved arginine is required for the transport of assembled receptors to the cell surface. By contrast, R209 does not play any role in the transport of assembled alpha-Bgtx-insensitive neuronal alpha3beta4 nAChRs to the cell surface. However, a basic residue at this position of alpha3 and beta4 subunits is necessary for either synthesis, folding, or assembly of alpha3beta4 receptors. Moreover, electrophysiological experiments revealed that in alpha3beta4 receptors the conserved arginine of the alpha3 subunit is involved in either coupling agonist binding to the channel or regulating single channel kinetics.

Multiple functional Sp1 domains in the minimal promoter region of the neuronal nicotinic receptor alpha5 subunit gene.

The alpha5 subunit is a component of the neuronal nicotinic acetylcholine receptors, which are probably involved in the activation step of the catecholamine secretion process in bovine adrenomedullary chromaffin cells. The promoter of the gene coding for this subunit was isolated, and its proximal region was characterized, revealing several GC boxes located close to the site of transcription initiation (from -111 to -40). Deletion analysis and transient transfections showed that a 266-base pair region (-111 to +155) gave rise to approximately 77 and 100% of the maximal transcriptional activity observed in chromaffin and SHSY-5Y neuroblastoma cells, respectively. Site-directed mutagenesis of five different GC motifs indicated that all of them contribute to the activity of the alpha5 gene, but in a different way, depending on the type of transfected cell. Thus, in SHSY-5Y cells, alteration of the most promoter-proximal of the GC boxes decreased alpha5 promoter activity by approximately 50%, whereas single mutations of the other GC boxes had no effect. In chromaffin cells, by contrast, modification of any of the GC boxes produced a similar decrease in promoter activity (50-69%). In both cell types, however, activity was almost abolished when four GC boxes were suppressed simultaneously. Electrophoretic mobility shift assays using nuclear extracts from either chromaffin or SHSY-5Y cells showed the specific binding of Sp1 protein to fragment -111 to -27. Binding of Sp1 to the GC boxes was also demonstrated by DNase I footprint analysis. This study suggests that the general transcription factor Sp1 plays a dominant role in alpha5 subunit expression, as has also been demonstrated previously for alpha3 and beta4 subunits. Since these three subunits have their genes tightly clustered and are expressed in chromaffin cells, probably as components of the same receptor subtype, we propose that Sp1 constitutes the key factor of a regulatory mechanism common to the three subunits.

Gating of alpha3beta4 neuronal nicotinic receptor can be controlled by the loop M2-M3 of both alpha3 and beta4 subunits.

Previous studies have shown that the gating mechanism of alpha3beta4 neuronal nicotinic receptors is affected by a residue in the middle of the M2-M3 loop of the beta4 subunit. We have extended the study of the same location to the alpha3 subunit. Bovine alpha3beta4 receptors were mutated in position 268, substituting the residue present in wild-type receptors, i.e. leucine in alpha3 and asparagine in beta4, for an aspartate. Wild-type and mutated alpha3 and beta4 subunits were combined to form four different receptors. We have measured macroscopic currents in Xenopus oocytes elicited by nicotine, and related them to surface receptor expression measured with an epibatidine-binding essay. We also obtained single-channel recordings of the receptors to study their kinetic behaviour. The results were analysed in terms of an allosteric model with three states. We found that the effect of the mutation in the alpha3 subunit on the gating of the receptor was similar to the corresponding mutation in the beta4 subunit. The effect when both subunits were mutated was additive, suggesting that the contribution of each subunit to the gating mechanism is independent.

A residue in the middle of the M2-M3 loop of the beta4 subunit specifically affects gating of neuronal nicotinic receptors.

An aspartate residue in the M2-M3 loop of neuronal nicotinic receptor alpha7 subunits is a major determinant of the channel functional response. This residue is conserved in most beta4 subunits, e.g. human and rat, but not in others, e.g. bovine. We have used these differences to examine the mechanism by which this residue alters the functional properties of alpha3beta4 receptors. Having ruled out an effect on the macroscopic binding ability of the agonist, the level of receptor expression, or the single channel conductance, the results suggest that receptors lacking that residue have a deficient coupling between binding and gating.

GC- and E-box motifs as regulatory elements in the proximal promoter region of the neuronal nicotinic receptor alpha7 subunit gene.

The alpha7 subunit is a component of alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors expressed in bovine adrenomedullary chromaffin cells. The proximal promoter of the gene coding for this subunit contains several GC-boxes and one E-box. Deletion analysis and transient transfections showed that a 120-base pair region (-77 to +43) including all of these elements gave rise to approximately 70 and 95% of the maximal transcriptional activity observed in chromaffin and SHSY-5Y neuroblastoma cells, respectively. Site-directed mutagenesis of the different elements indicated that both GC and E motifs contribute to the activity of the alpha7 gene in a very prominent way. Using electrophoretic mobility shift assays, the upstream stimulatory factor (USF) was shown to be a component of the complexes that interacted with the E-box when nuclear extracts from chromaffin and SHSY-5Y cells were used. Binding of the early growth response gene transcription factor (Egr-1) to three different GC-boxes was also demonstrated by shift assays and DNase I footprint analysis. Likewise, alpha7 promoter activity increased by up to 5-fold when alpha7 constructs and an Egr-1 expression vector were cotransfected into chromaffin cell cultures. Mutagenesis of individual GC-boxes had little effect on Egr-1 activation. By contrast, pairwise suppression of GC-boxes abolished activation, especially when the most promoter-proximal of the Egr-1 sites was removed. Taken together, these studies indicate that the alpha7 gene is likely to be a target for multiple signaling pathways, in which various regulatory elements are involved.

Role of the putative transmembrane segment M3 in gating of neuronal nicotinic receptors.

The involvement of some structural domains in the gating of the neuronal nicotinic acetylcholine receptor (AChR) was studied by expressing functional alpha7/alpha3 chimeric subunits in Xenopus oocytes. Substitution of the M3 transmembrane segment in the alpha7 subunit modifies the kinetic properties of the chimeric AChRs as follows: (a) a 6-fold reduction in the maximal current evoked by nicotinic agonists, (b) a 10-fold decrease in the macroscopic desensitization rate, (c) an increase of almost 1 order of magnitude in the apparent affinity for acetylcholine and nicotine, and (d) a decrease in the affinity for alpha-bungarotoxin. Computer simulations showed that the first three effects could be accounted for by a simple kinetic model in which chimeric AChRs presented a smaller ratio of the gating rates, beta/alpha, and a slightly slower desensitization rate. It is concluded that the M3 domain influences the gating of neuronal AChRs.

Neuronal nicotinic acetylcholine receptors on bovine chromaffin cells: cloning, expression, and genomic organization of receptor subunits.

Neuronal nicotinic acetylcholine receptors from bovine adrenomedullary chromaffin cells play a primary role in triggering catecholamine secretion. In the present study, their constituent subunits were characterized. In addition to the alpha 3 subunit, which we have previously cloned, the presence of alpha 5 and beta 4 but not of beta 2 subunits was detected by reverse transcription-PCR analysis of mRNA from adrenal medulla. In situ hybridization indicated that alpha 3, alpha 5, and beta 4 subunits are coexpressed in all chromaffin cells. The primary structure of alpha 5 and beta 4 subunits was determined and functional receptors were obtained upon coinjection of subunit cRNAs into Xenopus oocytes. In contrast to other beta 4-containing nicotinic receptors, the ones formed by the bovine beta 4 subunit are insensitive to the agonist cytisine. Finally, we characterized the intergenic region of alpha 3 and alpha 5 subunits, which together with the beta 4 subunit, form a gene cluster in rats and chickens. RNase assays and the existence of overlapping cDNAs indicate that, in the bovine genome, the alpha 3 and alpha 5 genes overlap at their 3' ends. This fact is probably due to inefficient transcription termination, as a result of weak polyadenylation signals.

Acetylcholine receptor subunit homomer formation requires compatibility between amino acid residues of the M1 and M2 transmembrane segments.

The neuronal nicotinic acetylcholine receptor (nAChR) subunits alpha3 and alpha7 have different assembly behavior when expressed in heterologous expression systems: alpha3 subunits require other subunits to assemble functional nAChRs, whereas alpha7 subunits can produce homomeric nAChRs. A previous analysis of alpha7/alpha3 chimeric constructs identified a domain comprising the first putative membrane-spanning segment, M1, as essential to homomeric assembly. The present study dissected further this domain, identifying three amino acid residues, which are located at the most intracellular third of the M1 transmembrane segment, as important in the assembly of homomers. Moreover, formation of homooligomeric complexes seems to require a compatible accommodation between this region and certain residues of the second transmembrane segment, M2. Thus, compatibility between defined domains of the M1 and M2 transmembrane segments appears as a determinant factor governing homomer association of nAChR subunits.

A single residue in the M2-M3 loop is a major determinant of coupling between binding and gating in neuronal nicotinic receptors.

Binding of agonists to nicotinic acetylcholine receptors generates a sequence of changes that activate a cation-selective conductance. By measuring electrophysiological responses in chimeric alpha7/alpha3 receptors expressed in Xenopus oocytes, we have showed the involvement of the M2-M3 loop in coupling agonist binding to the channel gate. An aspartate residue therein, Asp-266 in the alpha7 subunit, was identified by site-directed mutagenesis as crucial, since mutants at this position exhibited very poor functional responses to three different nicotinic agonists. We have extended this investigation to another neuronal nicotinic receptor (alpha3/beta4), and found that a homologous residue in the beta4 subunit, Asp-268, played a similar role in coupling. These findings are consistent with a hypothesis that the aspartate residue in the M2-M3 loop, which is conserved in all homomer-forming alpha-type subunits and all neuronal beta-type subunits that combine to form functional receptors, is a major determinant of information transmission from binding site to channel gate in all neuronal nicotinic receptors.

alpha-Bungarotoxin-sensitive nicotinic receptors on bovine chromaffin cells: molecular cloning, functional expression and alternative splicing of the alpha 7 subunit.

Chromaffin cells from the bovine adrenal medulla express alpha-bungarotoxin-sensitive acetylcholine receptors whose subunit composition is unknown. Northern blot analysis showed that the alpha 7 subunit, a main component of these alpha-bungarotoxin-sensitive acetylcholine receptors in avian and rat brain, is expressed in chromaffin cells. The cDNA of this bovine alpha 7 subunit was cloned by polymerase chain reaction amplification of adrenal medulla RNA for detailed characterization of structure and function. The protein-coding region revealed 92% amino acid sequence identity to rat alpha 7 and 89% to chicken alpha 7 subunits. The alpha-bungarotoxin affinity of alpha 7 homomers expressed in Xenopus oocytes was similar to that observed previously with native chromaffin alpha-bungarotoxin-sensitive acetylcholine receptors. Cross-linking and sucrose gradient experiments suggested that, like the muscular and neuronal acetylcholine receptors; the alpha 7 receptor has a pentameric structure. Upon activation with nicotinic agonists the alpha 7 receptor exhibited rapidly desensitizing cation currents that were blocked by nicotinic antagonists and showed inward rectification. The amplification of adrenal medulla RNA by reverse transcription-polymerase chain reaction methods revealed an alternatively spliced isoform of the bovine alpha 7 subunit, where the exon that codes for the M2 transmembrane segment was skipped during mRNA processing. Oocyte expression of this isoform does not yield functional channels. However, this alternative mRNA exhibits dose-dependent inhibition of alpha 7 homomer expression when coinjected with the undeleted isoform.

Role of two acetylcholine receptor subunit domains in homomer formation and intersubunit recognition, as revealed by alpha 3 and alpha 7 subunit chimeras.

Differential expression of subunit genes from the nicotinic acetylcholine receptor (AChR) superfamily yields distinct receptor subtypes. As each AChR subtype has a specific subunit composition and many subunit combinations appear not to be expressed, each subunit must contain some information leading to proper assembly. The neuronal AChR subunits alpha 3 and alpha 7 are expressed in bovine chromaffin cells, probably as constituents of two different AChR subtypes. These subunits have different assembly behavior when expressed in heterologous expression systems: alpha 7 subunits are able to produce homomeric AChRs, whereas alpha 3 subunits require other "structural" subunits for functional expression of AChRs. This feature allows the dissection of the requirements for subunit interactions during AChR formation. Analysis of alpha 7/alpha 3 chimeric constructs identified two regions essential to homomeric assembly and intersubunit recognition: an N-terminal extracellular region, controlling the initial association between subunits, and a second domain within a region comprising the first putative transmembrane segment, M1, and the cytoplasmic loop coupling it to the pore-forming segment, M2, involved in the subsequent interaction and stabilization of the oligomeric complex.