Human Alpha-subunit Research Articles

The multiphosphorylation domain of the phosphorylase kinase alpha M and alpha L subunits is a hotspot of differential mRNA processing and of molecular evolution.

We have cloned and sequenced human cDNAs encoding the complete phosphorylase kinase alpha subunit muscle isoform (alpha M). The predicted polypeptide is highly similar to the sequence known from rabbit muscle but lacks a major part of its multiphosphorylation domain, including the main phosphorylation site for cAMP-dependent protein kinase (PKA). Analysis of this region by reverse-transcribed polymerase chain reaction (RT-PCR) in several human and rabbit tissues demonstrates that it is subject to elaborate differential mRNA splicing. Amino acids 1012-1024 of the full-length rabbit sequence, including the major PKA phosphorylation site, and amino acids 1025-1041, which harbor at least one endogenous phosphorylation site, can be deleted from the predicted polypeptide individually or in combination. Molecules lacking one or both of these segments constitute a major part of the alpha M subunit population in many rabbit tissues and constitute the vast majority in all human tissues analyzed. Similar, tissue-dependent differential splicing events could be detected by RT-PCR in the human alpha subunit isoform from liver (alpha L). The expression of the differentially spliced alpha M subtypes differs markedly between corresponding human and rabbit tissues. Sequence divergence in this region is particularly high, not only between the muscle and liver isoforms, but also between alpha M sequences from four different animal species. Moreover, a duplication of the exon encoding the main PKA phosphorylation site was discovered in the mouse. Thus, the multiphosphorylation domain of the phosphorylase kinase alpha subunit isoforms is subject to pronounced structural variation not only between different tissues of one organism via differential splicing, but also in the course of evolution.

Estradiol modulates thyroid hormone regulation of the human glycoprotein hormone alpha subunit gene.

We have examined mechanisms of regulation of the human glycoprotein hormone alpha subunit gene by thyroid hormone (T3) and estradiol. Pituitary-derived GH3 cells were transiently transfected with chimeric constructs comprising between 1,500 and 98 base pairs of human alpha subunit gene 5'-flanking sequence fused to the bacterial gene encoding chloramphenicol acetyltransferase (h alpha CAT) and treated with T3 and estradiol, alone and in combination. In pituitary cells, 98 base pairs of alpha gene 5'-flanking sequence were sufficient to mediate both inhibition of alpha gene promoter activity by T3 and stimulation by estradiol; inhibition of the alpha promoter by T3 was antagonized by estradiol. Mutation of nucleotides essential for T3 receptor binding to the alpha gene thyroid hormone response element abolished the response of h alpha CAT expression to estradiol as well as T3. In contrast to pituitary GH3 cells, estradiol treatment alone had no effect on expression of either h alpha CAT or the endogenous alpha gene in JEG-3 choriocarcinoma cells cotransfected with a human thyroid hormone receptor expression vector, but estradiol antagonized suppression of both endogenous and transfected alpha promoter activity by T3. Gel mobility shift assays demonstrated specific binding of in vitro synthesized human estrogen receptor (ER) to the alpha gene thyroid hormone response element. These findings suggest that estradiol modulates expression of the human alpha subunit gene in pituitary and choriocarcinoma cells by direct binding of ER to the alpha gene promoter, and that interaction of ER with the alpha gene negative TRE accounts for the antagonistic effects of estradiol and T3.

CDNA Cloning of the Two Subunits of Human CAAX Farnesyltransferase and Chromosomal Mapping of FNTA and FNTB Loci and Related Sequences

The CAAX farnesyltransferase is a heterodimeric enzyme that attaches a farnesyl group to a single cysteine in several cellular proteins. Substrates include the p21ras proteins, nuclear lamins, and several retinal proteins, all of which end with a "CAAXbox," where C is cysteine, A is an aliphatic amino acid, and X is methionine or serine. Full-length cDNAs for the alpha and beta subunits of the rat farnesyltransferase have been cloned, and both have been shown to be essential for catalytic activity. Here we have used the rat cDNAs to clone cDNAs for the human alpha and beta subunits. Comparison of the human and rat amino acid sequences revealed a remarkable degree of conservation (93% identity for the alpha subunit and 96% identity for the beta subunit). The functional genes for the alpha and beta subunits of human farnesyltransferase (gene symbols, FNTA and FNTB) were localized to human chromosome bands 8p22-q11 and 14q23-q24, respectively, by Southern blot hybridization and PCR analyses of panels of human x Chinese hamster somatic cell hybrid lines and by fluorescence chromosomal in situ hybridization. We also found several related farnesyltransferase genes. FNTAL1 was assigned to 11q13.4-q14.1, FNTAL2 to chromosome 13, and FNTBL1 to chromosome 9.

Identification of sites on the human Fc epsilon RI alpha subunit which are involved in binding human and rat IgE.

The high affinity IgE Fc receptor (Fc epsilon RI), found on mast cells and basophils, is a tetrameric receptor complex. The extracellular portion of the Fc epsilon RI alpha subunit consists of two immunoglobulin-like domains and binds IgE in the absence of the other subunits. To localize the high affinity IgE binding site within the Fc epsilon RI alpha subunit, we generated a series of chimeric receptor constructs where one of the two immunoglobulin-like domains was either deleted or substituted with those from the human Fc gamma RIIIA alpha or the rat Fc epsilon RI alpha subunit. The chimeric receptors were monitored for their capacity to bind human and rat IgE, and their reactivity with different antireceptor antibodies. Domain I substitutions maintained high affinity human IgE binding. Domain II substitutions resulted in a total loss of both human and rat IgE binding. Single-domain alpha subunits could not bind IgE, suggesting that both extracellular domains are required for proper protein folding or IgE binding. To further localize the IgE binding sites, homolog-scanning mutagenesis was performed. At least three independent regions of domain II encompassing residues 118-129, 136-150, and 148-162 were required for IgE binding. Our results suggest that domain II of the human Fc epsilon RI alpha confers most of the important contributions to the binding of the human IgE Fc molecule, whereas domain I of the rat Fc epsilon RI alpha makes important contributions to the binding of rat IgE.

Site-directed alanine mutagenesis of Phe33, Arg35, and Arg42-Ser43-Lys44 in the human gonadotropin alpha-subunit.

Residues Phe33 and Arg35, individually, and a composite mutation of residues Arg42, Ser43, and Lys44 were changed to alanine in the human glycoprotein hormone common alpha-subunit using site-directed mutagenesis. These specific residues are highly conserved across species and have by chemical modification and synthetic peptide approaches been implicated in the binding of human chorionic gonadotropin (hCG) to leutinizing hormone (LH) receptor. In the present study we tested the hypothesis that specific alpha-subunit amino acid residues which stabilize the hormone receptor interaction for hCG have the same function in human follicle-stimulating hormone (hFSH). Wild type or mutant alpha-subunit cDNAs were coexpressed with wild type hFSH or hCG beta cDNA in sialylation defective Chinese hamster ovary cells. Recombinant hormones were tested in a radioligand receptor competition assay, using rat testis membranes as a source of FSH and LH receptors. Mutant hFSH heterodimers F33A-FSH, R35A-FSH, Arg42-Ser43-Lys44/Ala42-Ala43-Ala44- FSH all displaced 125I-hFSH in a similar fashion, indicating that these residues are not important for binding of hFSH to the rat FSH receptor. On the other hand, F33A-CG evidenced a 5-fold decrease in binding, while R35A-CG had over a 100-fold decrease in binding to the rat LH receptor when compared to the wild type recombinant hCG. These data demonstrate that a receptor-binding site on the common alpha-subunit which is very important for hCG binding to LH receptor is not important for the binding of hFSH to FSH receptor. Our interpretation of these findings is that there are fundamental structural differences in the receptor interface contacts of the common alpha-subunit, which stabilize receptor binding among members of the glycoprotein hormone family.

Effects of subunit mutation on the localization to coated pits and internalization of cross-linked IgE-receptor complexes.

IgE receptors of mast cells, Fc epsilon RI, localize to coated pits and internalize after cross-linking. We investigated whether any one of the receptor's four distinctive cytoplasmic domains regulates these phenomena. COS cells, which lack Fc epsilon RI entirely, and P815 mouse mastocytoma cells that lack the alpha and beta subunits of the tetrameric Fc epsilon RI (alpha beta gamma 2), were transfected with wild-type, incomplete, or variant Fc epsilon RI. IgE-receptor complexes were observed by electron microscopy. Before cross-linking with anti-IgE gold particles, receptors were not preferentially localized to coated pits, which occupy approximately 1% of the cell surface. After cross-linking, up to 10 to 20% of the wild-type and most other receptor variants were in coated pits in transfected P815 cells at any one time. beta-less variants localized normally but, surprisingly, receptors containing a variant beta subunit showed reduced localization. "Receptors" consisting simply of the lipid-anchored ectodomains of the human alpha subunit failed to localize to coated pits. In general, cross-linked receptors that localized to coated pits were progressively internalized, whereas receptors that failed to accumulate in coated pits were not. We conclude that no single cytoplasmic domain of the Fc epsilon RI uniquely controls its ligand-induced localization to coated pits and internalization.

Recombinant Human Thyroid Stimulating Hormone: Development of a Biotechnology Product for Detection of Metastatic Lesions of Thyroid Carcinoma

We have genetically engineered a cell line, and developed a reproducible process, for the expression and purification of biologically active recombinant human thyroid stimulating hormone (rhTSH).rhTSH was expressed by co-transfecting a human alpha-subunit cDNA with a human beta-subunit partial genomic clone into Chinese Hamster Ovary (CHO) cells. Stable transfectants which expressed high levels of rhTSH were selected, and subsequently cultured on microcarrier beads. The rhTSH-containing media, produced under serum-free conditions, was clarified and purified by a combination of ion exchange, dye and gel filtration chromatographies. Individual step recoveries were greater than 90% with the exception of a very conservative pooling of the final gel filtration step (78% recovery) that resulted in a cumulative yield of 54% for the purification process. Purity of the final bulk material was judged to be > 99% by SDS polyacrylamide gel electrophoresis (SDS-PAGE), reverse phase HPLC, and size exclusion chromatography. Initial characterization of the oligosaccharide composition indicated the presence of partially sialylated bi- and triantenary complex oligosaccharides. Purified rhTSH was active in a thyroid membrane bioactivity assay with a specific activity of 8.2 IU/mg. The in vivo activity of rhTSH in cynomolgus monkeys appeared to be equal to or greater than that reported for bovine TSH (bTSH) in human subjects. The rapid clearance phase half-life of rhTSH was approximately 35 minutes while the post-distribution phase half life was approximately 9.8 hours. Furthermore, the monkeys showed cumulative increases in minimum plasma rhTSH levels when given three daily intramuscular (IM) rhTSH injections; a phenomenon not observed when bTSH had been administered to humans. The rhTSH showed no evidence of toxic or adverse effects when administered at doses up to 7.2 IU/kg and 0.52 IU/kg in rat and monkey, respectively. These are 50X and 4X multiples of the bTSH doses of 0.143 IU/kg (10 IU/70kg) previously administered to humans.

Primary structure of the human muscle acetylcholine receptor. cDNA cloning of the gamma and epsilon subunits.

cDNA sequences encompassing the full coding region for the human muscle acetylcholine receptor (AChR) epsilon and gamma subunits have been isolated. The deduced amino-acid sequences indicate that the mature epsilon subunit contains 473 amino acids and is preceded by a 20-amino-acid signal peptide. As predicted from genomic clones, the gamma subunit contains 495 amino acids preceded by a 22-amino-acid signal peptide. In common with the human alpha, beta, gamma and delta subunits the epsilon subunit is highly conserved between mammalian species. The epsilon subunit gene is not closely linked to the gamma and delta subunits on chromosome 2 but rather is located with the beta subunit on chromosome 17. Expression of the alpha-, beta-, gamma-, delta- and epsilon-subunit cRNAs in rabbit-reticulocyte lysates followed by analysis on SDS/PAGE show glycosylated proteins with apparent molecular masses of 44-60 kDa.

Unexpected Effects of the IL-2 receptor alpha subunit on high affinity IL-2 receptor assembly and function detected with a mutant IL-2 analog

Binding of IL-2 to the high affinity IL-2R results in the formation of a stable complex consisting of IL-2 and at least three distinct, noncovalently associated receptor subunits: IL-2R alpha (p55), IL-2R beta (p70-75), and IL-2R gamma (p64). Ligand binding also stimulates the rapid endocytosis of this receptor complex. To gain further insight into the function of the various subunits of the multicomponent high affinity IL-2R complex, we have carried out binding, internalization, and proliferation assays using an IL-2 analog designated F42A that contains a single Phe for Ala amino acid substitution at position 42. This mutation markedly reduces the intrinsic affinity of the resultant IL-2 analog for the low affinity IL-2R alpha subunit although having little or no effect on binding to the IL-2R beta (or IL-2R beta/gamma) intermediate affinity receptor. We have confirmed that F42A does not bind to the IL-2R alpha chain when expressed alone on MT-1 cells or in the presence of the large or small excess of IL-2R beta chains present on either YT-1 cells or forskolin-induced YT-1 cells, respectively. However, although F42A does not interact with the large number of low affinity IL-2R alpha chains present on HUT 102B2 cells, this ligand does bind to the small number of IL-2R beta chains present on these cells with at least 10-fold higher than expected affinity. These findings indicate that excess IL-2R alpha chains may exert positive effects on binding perhaps by changing the conformation of IL-2R beta. In F42A-stimulated internalization assays on forskolin-induced YT-1 cells, the IL-2R alpha chain is consistently endocytosed together with the IL-2R beta subunit indicating that IL-2R alpha is stably associated with the F42A-IL-2R beta complex even though the alpha-subunit contributes little if any affinity to the F42A binding reaction. In proliferation assays on mouse IL-3-dependent pro-B BA/F3 cells stably coexpressing transfected human IL-2R alpha and IL-2R beta subunits (but a mouse IL-2R gamma subunit), F42A proved to be a more effective agonist of growth than wild-type IL-2. These results suggest that the interaction between wild-type IL-2 but not F42A and the IL-2R alpha subunit in this mixed species high affinity receptor complex may induce an unfavorable receptor conformation leading to diminished rather than enhanced growth signal transduction.

Transcriptional repression of the glycoprotein hormone alpha subunit gene by androgen may involve direct binding of androgen receptor to the proximal promoter

Testicular androgens suppress the synthesis and secretion of the pituitary gonadotropins, in particular, luteinizing hormone. This suppressive effect includes transcription of both the common alpha subunit gene and the unique beta subunit genes. Herein, we demonstrate that 1500 base pairs (bp) of proximal 5'-flanking region derived from the human alpha subunit gene and a shorter 315-bp segment of the bovine alpha subunit gene confer negative regulation by androgen to the gene encoding bacterial chloramphenicol acetyltransferase in transgenic mice. Cotransfection assays with human androgen receptor indicated that the 1500-bp promoter region of the human alpha subunit gene also confers androgen regulation (transcriptional suppression) to reporter genes in both pituitary and placental cell lines. This raises the possibility of a role for DNA binding in suppression of alpha subunit transcription by activated androgen receptor. Consistent with this possibility, we have used a gel-mobility shift assay to detect several high affinity binding sites for androgen receptor located in the proximal promoter of the human alpha subunit gene. The strongest androgen receptor binding site is located at approximately -101 in the proximal 5'-flanking region. This steroid receptor binding site overlaps another binding site that defines one of several contiguous cis-acting regulatory elements required for basal transcriptional activity. Thus, binding of activated androgen receptor to this region may block the binding of a requisite trans-acting factor and lead to an attenuation in transcription. We conclude that this interaction, which occurs directly at the level of the pituitary, represents one of several physiological avenues through which androgens regulate gonadotropin gene expression.

Phage and Escherichia coli expression of the human high affinity immunoglobulin E receptor alpha-subunit ectodomain. Domain localization of the IgE-binding site

The high affinity IgE receptor is a multisubunit complex that participates in IgE-dependent activation of mast cells and basophils. The IgE-binding portion of the receptor resides exclusively in the alpha-subunit and specifically within the 180 residues of the mature extracytoplasmic portion. In this study the contiguous two-domain human alpha-subunit has been displayed on the surface of a filamentous bacteriophage in a form that specifically binds both human and murine IgE but not other isotypes. Phage display of the individual immunoglobulin-like domains reveals that the IgE-binding portion resides exclusively in the COOH-terminal domain and that this domain appears to bind IgE with lower affinity than the comparably displayed two-domain fragment. Phage display results using a chimeric rat-human alpha-subunit fragment suggest that structural elements within the NH2-terminal domain are necessary to impart high affinity IgE binding activity to the alpha-chain ectodomain. The two-domain human receptor fragment was also expressed in a soluble form from Escherichia coli and purified in one step by affinity chromatography. The solution binding of the purified receptor fragment to IgE was measured by fluorescence quench which afforded an approximate equilibrium affinity constant of 2 x 10(9) M-1 together with a stoichiometry of 1 receptor molecule/molecule of IgE. The complementary approach of phage display and E. coli expression used in this study represents a useful strategy for further analysis of discrete receptor epitope(s) that contribute to IgE binding activity.

Unexpected effects of the IL-2 receptor alpha subunit on high affinity IL-2 receptor assembly and function detected with a mutant IL-2 analog.

Abstract Binding of IL-2 to the high affinity IL-2R results in the formation of a stable complex consisting of IL-2 and at least three distinct, noncovalently associated receptor subunits: IL-2R alpha (p55), IL-2R beta (p70-75), and IL-2R gamma (p64). Ligand binding also stimulates the rapid endocytosis of this receptor complex. To gain further insight into the function of the various subunits of the multicomponent high affinity IL-2R complex, we have carried out binding, internalization, and proliferation assays using an IL-2 analog designated F42A that contains a single Phe for Ala amino acid substitution at position 42. This mutation markedly reduces the intrinsic affinity of the resultant IL-2 analog for the low affinity IL-2R alpha subunit although having little or no effect on binding to the IL-2R beta (or IL-2R beta/gamma) intermediate affinity receptor. We have confirmed that F42A does not bind to the IL-2R alpha chain when expressed alone on MT-1 cells or in the presence of the large or small excess of IL-2R beta chains present on either YT-1 cells or forskolin-induced YT-1 cells, respectively. However, although F42A does not interact with the large number of low affinity IL-2R alpha chains present on HUT 102B2 cells, this ligand does bind to the small number of IL-2R beta chains present on these cells with at least 10-fold higher than expected affinity. These findings indicate that excess IL-2R alpha chains may exert positive effects on binding perhaps by changing the conformation of IL-2R beta. In F42A-stimulated internalization assays on forskolin-induced YT-1 cells, the IL-2R alpha chain is consistently endocytosed together with the IL-2R beta subunit indicating that IL-2R alpha is stably associated with the F42A-IL-2R beta complex even though the alpha-subunit contributes little if any affinity to the F42A binding reaction. In proliferation assays on mouse IL-3-dependent pro-B BA/F3 cells stably coexpressing transfected human IL-2R alpha and IL-2R beta subunits (but a mouse IL-2R gamma subunit), F42A proved to be a more effective agonist of growth than wild-type IL-2. These results suggest that the interaction between wild-type IL-2 but not F42A and the IL-2R alpha subunit in this mixed species high affinity receptor complex may induce an unfavorable receptor conformation leading to diminished rather than enhanced growth signal transduction.

An enzyme-linked immunoadsorbent assay for human pituitary glycoprotein free alpha-subunit: evaluation and initial experience in patients with pituitary disease.

An enzyme-linked immunoadsorbent assay (ELISA) for measuring human pituitary glycoprotein free alpha-subunit is described. Pituitary tumours may secrete glycoprotein hormones, their free subunits (alpha, beta) or a combination of these. Non-functioning adenomas often secrete free alpha-subunit. Assays for free alpha-subunit have previously used radioimmunoassay or immunoradiometric principles. Some of these methods are time-consuming and lack specificity and sensitivity. In order to overcome these problems, we have developed a two-site ELISA for alpha-subunit which uses a monoclonal antibody to alpha-subunit as the capture antibody to provide greater specificity. An affinity-purified polyclonal anti alpha-subunit conjugated to alkaline phosphatase was the signal antibody. Detection and quantification were based on phenolphthalein monophosphate conversion to phenolphthalein. The ELISA specifically measured glycoprotein free alpha-subunit in serum or plasma, discriminating between alpha-subunit and the intact glycoprotein hormones. The assay can be completed in 4 h. The assay sensitivity was 0.03 micrograms/l, and a normal range of 0.05 to 0.22 micrograms/l was established. In a retrospective study, elevated circulating glycoprotein alpha-subunit was detected in 22% of patients with non-functioning pituitary tumours previously treated with surgery and/or radiotherapy.

Ia-antigen-T-cell interactions for a thymus-independent antigen composed of D amino acids.

Synthetic polypeptide antigens of L amino acids, although bearing repeating sequences, are thymus-dependent (L-TD), whereas the same polymers composed of D amino acids are thymus-independent (D-TI), probably due to a slower rate of metabolism. Yet we found that lymph-node cells of BALB/c mice immunized with D-TI proliferate in response to it in vitro. To follow T-cell activation by D-TI, we established T-cell hybridomas to D-TI and to its analog composed of L isomers, L-TD, for comparison. The T-cell hybridomas express membrane alpha/beta T-cell receptors and secrete interleukin 2 upon stimulation with the respective antigen. In addition, D-TI-specific hybridomas are stimulated, to a lesser extent, by the L-TD antigen, whereas only some L-TD-specific hybridomas recognize D-TI. Moreover, biotinylated analogs of D-TI and L-TD bind to splenic antigen-presenting cells (APCs) from BALB/c mice. Binding is inhibited by an excess of nonbiotinylated L-TD, and by an excess of a peptide comprising residues 259-271 of the human acetylcholine receptor alpha subunit, which binds to I-Ad and I-Ed molecules without prior processing. Analysis of APC lysates following incubation of the APCs with biotinylated D-TI and L-TD reveals that the biotinylated antigen moiety is associated with Ia molecules. D-TI and L-TD bind to Ia molecules on intact APCs with similar KD values, 5 x 10(-8) M and 3 x 10(-8) M, respectively. However, D-TI has faster kinetics of binding than L-TD, probably due to different processing requirements. Hence, we have demonstrated a major histocompatibility complex class II-mediated T-cell response to a thymus-independent antigen.

Soluble human high-affinity receptor for IgE abrogates the IgE-mediated allergic reaction.

The high-affinity receptor for IgE (Fc epsilon RI) has a tetrameric structure structure composed of one alpha, one beta, and two disulfide-linked gamma subunits, of which the alpha subunit binds IgE with high affinity. A recombinant soluble form of the ectodomain of the human Fc epsilon RI alpha subunit (rsFc epsilon RI alpha) was recently generated by gene engineering and was verified to bind IgE with an affinity as high as that of native Fc epsilon RI on the cell surface. rsFc epsilon RI alpha was prepared on a large scale in order to analyze its biological function. rsFc epsilon RI alpha completely inhibited IgE binding to the cell surface, resulting in abrogation of the chemical mediator release from RBL-2H3 cells. Furthermore it completely abolished the passive cutaneous anaphylaxis (PCA) response by trapping IgE specifically when it was administered into rats prior to IgE sensitization. Even after IgE sensitization, treatment of rsFc epsilon RI alpha substantially reduced the PCA response. It was finally shown that rsFc epsilon RI alpha inhibited IgE binding to human peripheral blood basophils and the histamine release from them. In this paper we address the ability of rsFc epsilon RI alpha to specifically prevent the IgE-mediated allergic reaction.

Mapping of a liver phosphorylase kinase alpha-subunit gene on the mouse X chromosome.

Phosphorylase kinase (PHK) is a regulatory enzyme of the glycogenolytic pathway composed of a complex of four subunits. We recently mapped the muscle alpha-subunit gene (Phka) to the mouse X chromosome in a region syntenic with the proximal long arm of the human X chromosome and containing the human homologue of this gene, PHKA. We now report the mapping of the liver alpha-subunit gene to the telomeric end of the mouse X chromosome. This mapping position would suggest a location for the human liver alpha-subunit gene on the proximal short arm of the X chromosome, a region recently implicated in X-linked liver glycogenosis (XLG).

Myasthenia gravis: effect on antibody binding of conservative substitutions of amino acid residues forming the main immunogenic region of the nicotinic acetylcholine receptor.

In Myasthenia Gravis most anti-acetylcholine receptor (AChR) antibodies are against a highly conserved area of the AChR alpha-subunit called the Main Immunogenic Region (MIR). Amino acid residues critical for MIR formation have been located within the sequence alpha 67-76. In the present study, binding of anti-AChR monoclonal antibodies (mAbs) to synthetic peptide analogues of the sequence alpha 67-76 of human and Torpedo AChRs containing conservative single-residue substitutions identified the amino acid residues most important to the antigenicity of the MIR sequence, and offered clues to its tridimensional structure. Conservative substitutions of residues Asn68 and Asp71 greatly diminished mAb binding, identifying them as critical contact residues for anti-MIR mAbs. Substitutions at Asp70 and Tyr72 moderately affected binding. Cross-reactive mAbs originally raised against Electrophorus AChR bound single residue-substituted synthetic peptides in a manner consistent with the possibility that Electrophorus AChR may have a glutamic acid residue at position alpha 70 or alpha 71. Substitutions at residues Asp/Ala70 and Val/Ile70 between human and Torpedo alpha-subunits may be size-compensating, suggesting these amino acids in the native AChR may be in closer proximity than proposed in previous models of the MIR.

Reduction and reoxidation of equine gonadotropin alpha-subunits

Ovine (o) and equine (e) LH alpha-subunits were reduced and reoxidized using conditions known to be effective for bovine and human alpha-subunits. The major product of oLH alpha refolding was alpha-subunit monomer. In contrast, eLH alpha formed a 121,000 mol wt aggregate. Monomeric eLH alpha was recovered, but in greatly reduced yield. To test the effects of carbohydrate variation on the aggregation of equine alpha-subunits, all of the equine gonadotropin alpha-subunits (eFSH alpha, eCG alpha, eLH alpha, and free alpha-subunit) were reduced and reoxidized. In each case, the major product was the 121,000 mol wt aggregate accompanied by monomeric equine alpha. Removal of carbohydrate by trifluoromethane sulfonic acid hydrolysis accentuated the tendency to aggregation during reoxidation. Most reduced-reoxidized deglycosylated eLH alpha did not enter a 12% sodium dodecyl sulfate-polyacrylamide gel. The highest LH receptor-binding activities were found in the alpha-subunit preparations, eLH alpha itself and pi...

Subunit folding and alpha delta heterodimer formation in the assembly of the nicotinic acetylcholine receptor. Comparison of the mouse and human alpha subunits

We have used the mouse alpha (alpha M) and human alpha (alpha H) subunits to investigate the molecular mechanisms of assembly of the mammalian acetylcholine receptor (AChR) transiently expressed in COS cells. COS cells expressing hybrid receptors incorporating alpha H along with other mouse subunits exhibited a 2-fold higher level of surface alpha-bungarotoxin (BuTx) binding than cells expressing the wild-type mouse AChR. When expressed either alone or with the delta subunit in COS cells, alpha H acquired the BuTx binding conformation (alpha Tx) more efficiently than did alpha M. By oligonucleotide-directed mutagenesis we showed that 2 residues in the amino-terminal domain were responsible for the differences between alpha M and alpha H. Alpha MST, the modified mouse alpha subunit, both folded more efficiently to form alpha Tx and was more effective in forming a stable alpha delta heterodimer than was alpha M. The kinetics of alpha Tx and alpha delta heterodimer formation revealed that the delta subunit increased the conversion of immature forms of the alpha subunit into the BuTx binding form and therefore provides evidence for interaction between the delta subunit and the immature form of the alpha subunit. These results provide evidence of the importance of the amino-terminal domains of the AChR subunits in the assembly process.

Identification of a gonadotropin-releasing hormone-responsive region in the glycoprotein hormone alpha-subunit promoter.

Transcriptional regulation of the glycoprotein hormone alpha-subunit gene has been studied extensively in placental cells, but much less is known about its regulation in the pituitary gland. In this study, transcriptional control of the human alpha-subunit gene by GnRH was analyzed using transient expression assays in primary cultures of rat pituitary cells using alpha promoter constructs linked to a luciferase reporter gene. Deletion mutants between -846 and -156 basepairs (bp) had little effect on basal expression, but further deletion to -132 bp reduced basal activity by approximately 50%. Deletion of a cAMP response element between -132 and -99 bp caused a marked loss of basal activity, reducing expression to that of background luciferase activity. The same constructs were analyzed for cAMP responsiveness in primary pituitary cells. The degree of stimulation with 1 mM 8-bromo-cAMP (3.6- to 6.0-fold) was relatively unaffected by deletions from -846 to -132 bp, whereas cAMP stimulation was decreased by further deletion to -99 bp, consistent with the presence of previously defined cAMP response elements in this region of the alpha promoter. GnRH stimulation of alpha promoter activity was highly dependent upon the time of hormone addition after transfection, being most effective when added soon after transfection. Under optimal conditions, GnRH stimulated -846 alpha LUC expression by 20-fold. GnRH responsiveness was retained with deletion to -346 bp, but it was decreased by 55% after deletion to -280 bp and by 79% with deletion to -244 bp.(ABSTRACT TRUNCATED AT 250 WORDS)