Spectrin is associated with membrane-bound actin filaments in platelets and is hydrolyzed by the Ca2+-dependent protease during platelet activation

We recently showed that platelets contain submembranous actin filaments that are linked to glycoprotein (GP) Ib on the plasma membrane. In the present study, experiments were performed to determine whether spectrin was associated with these filaments. The membrane-bound filaments were isolated from Triton X-100 (Sigma, St Louis) lysates of unstimulated platelets by differential centrifugation. Platelet spectrin was detected immunologically by using antibodies against human brain and RBC spectrin. Immunoblots showed that platelet spectrin consisted of two polypeptides (mol wt 240,000 and 235,000) that were similar in apparent mol wt to those of the alpha and beta chains of brain spectrin but differed slightly from those of RBC spectrin (mol wt 240,000 and 220,000). Immunoprecipitation experiments identified platelet spectrin as two minor polypeptides migrating on sodium dodecyl sulfate (SDS)- polyacrylamide gels between actin-binding protein (mol wt 250,000) and the platelet polypeptide P235 (mol wt 235,000). Immunoblots of fractions isolated from Triton X-100-lysed platelets revealed that the alpha and beta chains of platelet spectrin were associated almost entirely with the actin filaments that were linked to the plasma membrane. Little spectrin was recovered in the Triton X-100-soluble fraction or with the actin filaments that were not membrane bound. During activation of platelets with thrombin or ionophore A23187, the alpha and beta chains of spectrin were hydrolyzed, generating a major degradation product of mol wt 160,000 and a minor one of mol wt 170,000. These two hydrolytic products were also generated in Triton X- 100 lysates incubated in the presence of Ca2+ but were not produced when lysates were treated with leupeptin, ethylene glycol bis(beta- aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), or N- ethylmaleimide, known inhibitors of the Ca2+-dependent protease. These experiments show that spectrin is a previously unidentified component of the membrane-bound actin filament network and that hydrolysis of spectrin by the Ca2+-dependent protease may regulate the interactions of the filaments during platelet activation.     

Identification of a spectrin-like protein in nonerythroid cells.

We have demonstrated the existence of a spectrin-like protein in a variety of nonerythroid cultured cells. Indirect immunofluorescence studies with monospecific antispectrin IgG indicated the presence of proteins that have common antigenic determinants to spectrin in embryonic chicken cardiac myocytes, mouse fibroblast lines (3T3, simian virus 4-transformed 3T3), and rat hepatoma lines (HTC, HMOA). Two spectrin-like peptides of 240,000 and 230,000 daltons were immunoprecipitated from octyl glucoside-solubilized embryonic chicken cardiac myocytes, along with associated cytoskeletal proteins. Immunoautoradiographic characterization of the myocyte immunoprecipitate showed that only the spectrin-like 240,000- and 230,000-dalton peptides were stained with monospecific antispectrin IgG and 125I-labeled protein A. One-dimensional partial proteolytic mapping of the myocyte 240,000- and 230,000-dalton peptides showed that these peptides share substantial sequence homology with embryonic chicken erythrocyte spectrin 240,000- and 220,000-dalton peptides.     

Calmodulin-binding proteins in plasma membranes from adrenocortical cells

Highly purified plasma membranes from Y-1 mouse adrenal tumor cells and those from bovine fasciculata cells were shown by [125I]iodocalmodulin overlay to contain five calmodulin-binding proteins of 240,000, 150,000, 66,000, 60,000, and 51,000 mol wt (Mr). Three of these proteins were also detected by affinity chromatography on calmodulin-Sepharose. Calmodulin binding was inhibited by competition with unlabeled calmodulin and by an inhibitor of calmodulin (trifluoperazine). Binding to each of the proteins was Ca2+ dependent. The relative proportion of binding to each of the five proteins was very different for Y-1 and bovine membranes. In Y-1 membranes as much as 50% of total binding was to the 51,000 Mr protein, whereas in bovine membranes more than 50% of binding occurred with the 150,000 Mr protein. Three of the five proteins were tentatively identified as follows: the 240,000 Mr protein is alpha-spectrin, the 60,000 Mr protein is the A subunit of the Ca2+/calmodulin-dependent protein phosphatase called calcineurin and the 51,000 Mr protein is the major subunit of a Ca2+/calmodulin-dependent protein kinase. The kinase was shown to act on specific substrates. It is concluded that calmodulin, by binding to the kinase and phosphatase, is capable of influencing the degree of phosphorylation of specific substrates in the plasma membranes of adrenal cells, and by binding to alpha-spectrin it may influence the cytoskeletons of these cells. These effects of calmodulin are likely to be important in the regulation of steroid synthesis in the adrenal cortex.     

Estradiol has inverse effects on pituitary glycoprotein hormone alpha-subunit messenger ribonucleic acid in the immature European eel and the gonadectomized rat

In teleosts, the pituitary contains a single glycoprotein gonadotropic hormone (GTH) composed of two dissimilar alpha- and beta-subunits. The European eel, Anguilla anguilla L, is sexually immature at the silver stage due to a deficiency in GTH synthesis and secretion. In previous studies we (S.D., YA.F.) have demonstrated a strong stimulatory action of estradiol (E2) on eel pituitary GTH content. In contrast, we (R.C., M.J.) have shown that in the rat E2 negatively regulates gonadotropin subunit synthesis via changes in specific mRNA levels. The purpose of our present work was to check for such effects of E2 on the synthesis of GTH alpha- and beta-subunits in the European eel. Eel pituitary mRNA was translated in a cell-free system in the presence of [35S]Met + Cys. We demonstrate that one of the translated polypeptides, characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, cross-reacts with an antiserum to denatured bovine alpha-subunit. Its apparent mol wt (18.5K), which is slightly higher than that of the corresponding rat alpha-precursor, suggests that it represents the precursor of the alpha-subunit of eel glycoprotein hormones. The specificity of immunoprecipitation was confirmed by competition with ovine alpha (but not with ovine LH beta or bovine TSH beta). Quantitative evaluation of the putative eel alpha-subunit precursor showed that it represents 0.2% of the total protein translated by RNA from the normal silver eel. Chronic treatment of eels for 3 weeks with 17 beta-E2 increased by 8.0- to 8.5-fold the proportion of the putative alpha-subunit precursor among translation products. Due to the lack of cross-reactivity with the presumed GTH beta precursor, no radioactive material could be specifically detected in translation medium of eel pituitary mRNA using antisera to either denatured bovine LH beta or ovine FSH beta. Our data suggest that E2, depending on vertebrate group and probably on sexual status, may exert either positive or negative control on gonadotropin synthesis by opposite effects on the levels of specific mRNA.     

Normal content of brain spectrin-like protein in sph/sph mice

In the erythrocytes of WBB6F1- sph/sph mice spectrin constitutes only - 1% of the total sph/sph membrane protein compared to - 23% in WBB6F1-+/+ controls. No increase in proteolytic degradation of spectrin in sph/sph erythrocyte membranes could be detected with antibodies directed againt mouse erythrocyte spectrin or mouse brain spectrin-like protein. As attachment of normal spectrin to the erythrocyte membrane of these animals appeared to be normal, and as spectrin not detected when whole sph/sph erythrocytes are solubilized in SDS for SDS PAGE, the deficient erythrocyte spectrin was probably due to diminished production. Brain spectrin-like protein, a nonerythroid spectrin analogue, is antigenically, morphologically and functionally related to erythrocyte spectrin, but appears by peptide mapping analysis to be a distinct gene product. It was found by protein- and antibody-staining of brain membranes to be present in normal concentrations in sph/sph animals. Indirect immunofluorescence of mouse brain tissue with anti-brain spectrin-like protein IgG or anti-erythrocyte spectrin IgG indicated that the distribution of brain spectrin-like protein was normal in sph/sph brain. Therefore the mutation causing diminished production of sph/sph erythrocyte spectrin does not affect the expression of this nonerythroid spectrin analogue.     

Involvement of spectrin in cell-surface receptor capping in lymphocytes.

Human and mouse lymphocytes of T- and B-cell lineages express a protein (Mr, 240,000) that crossreacts with antibodies raised against chicken erythrocyte alpha-spectrin as judged by immunofluorescence, immunoprecipitation, and immunoautoradiography; by the same criteria, antibodies raised against chicken erythrocyte beta-spectrin do not react with any lymphocyte polypeptide. In all T and B cells analyzed, before surface-directed ligand challenge with concanavalin A and surface immunoglobulins the polypeptide antigenically related to erythrocyte alpha-spectrin is distributed diffusely at the plasma membrane. Upon challenge, the redistribution of this polypeptide is concurrent with that of the cell-surface receptors initially in patches and then in a cap. Immunoprecipitation of NaDodSO4-solubilized lymphocytes with erythrocyte alpha-spectrin antiserum shows that in all cases a polypeptide with the same apparent molecular weight as erythrocyte alpha-spectrin is precipitated. Variable amounts of another polypeptide (Mr, 235,000) are also coimmunoprecipitated. Immunoprecipitations and subsequent immunoautoradiography show that the lymphocyte polypeptide doublet has a composition similar to that of (brain) fodrin, a polypeptide doublet that previously has been found mainly in the cells of nervous tissue.     

Differential production and regulation of inhibin subunits in rat testicular cell types

The distributions of the alpha-, beta A-, and beta B subunits of inhibin/activin polypeptides were studied in the testis of adult (60-day-old) and immature (12-day-old) rats. Immunohistochemical techniques using antisera selective for each subunit were used to localize the polypeptide chains. In situ hybridization using radiolabeled complementary RNA probes enabled localization of the messenger RNAs (mRNAs) encoding these subunits. In 12-day-old rats, immunostaining and mRNA signal for the alpha-subunit was found in Leydig cell clusters. The beta A- and beta B-subunit staining and beta A-subunit message were detectable in isolated interstitial cells, but the clusters appeared to lack these subunits. Positive immunostaining for each subunit was localized in a Sertoli cell-like pattern in seminiferous tubules, as was a positive mRNA signal for the alpha- and beta B-subunit over regions containing these cell types. Treatment with human CG (hCG) and PMSG greatly enhanced the production of the alpha-subunit in the Leydig cell clusters, but not within the tubules, of these young rats. In adult rats, alpha- and beta B-subunit staining, and alpha-subunit mRNA signal, was observed in the interstitial cells. As in the immature animals, all three subunits were localized in a Sertoli cell-like pattern in the tubules, and a positive mRNA signal for the alpha- and beta B-subunits was found over these cells. There was, however, no obvious change in the expression of the subunits in the testis of adult rats after gonadotropin treatment. The present findings suggest that: 1) in the rat testis, both Sertoli and interstitial cells produce inhibin/activin subunits; 2) the alpha- and beta-subunits are produced by different types of interstitial cells in immature rats; and 3) the production of the alpha-subunit in the Leydig cells of immature rats is regulated by LH-like hormones.     

Characterization of the subunit structure of the thyrotropin receptor in the FRTL-5 rat thyroid cell line

Radioiodinated TSH was covalently cross-linked to monolayers of FRTL-5 rat thyroid cells using the homobifunctional cross-linking agent disuccinimidyl suberate. Analysis of the cross-linked samples by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions demonstrated the presence of three specifically labeled complexes with apparent mol wt of 68,000, 85,000, and 145,000, in addition to the TSH alpha-beta dimer and its alpha- and beta-subunits. When bound [125I]iodo-TSH was cross-linked with increasing concentrations of disuccinimidyl suberate, the formation of the 68,000 and 85,000 mol wt complexes was sequential, with the 68,000 complex appearing first. These two complexes were also observed after labeling with radioiodinated TSH hybrid molecules (alpha-beta or alpha-beta), in which the label is in only one subunit, or immuno-precipitation with antibodies against either the alpha- or beta-subunit of TSH. Similar complexes (65,000, 82,000, and 145,000 mol wt) were also formed after cross-linking with the alkaline-cleavable cross-linker (bis-[2-(succinimidooxycarbonyloxy)ethyl]sulfone. Again, the appearance of these three complexes was sequential and dependent on the cross-linker concentration. At low concentrations and under reducing conditions, the 65,000 mol wt complex was the major band. However, at high concentrations, especially under nonreducing conditions, most of the radioactivity was present in the 145,000 mol wt complex. Alkaline cleavage of these three complexes followed by electrophoresis in a second dimension resulted in the release of three components with approximate mol wt of 31,000, 17,000, and 63,000 in addition to the TSH alpha-beta dimer and its alpha- and beta-subunits. Reduction by dithiothreitol followed by electrophoresis in a second dimension resulted in the release of only the 17,000 and 63,000 components. Taken together, these results suggest that 1) both the 65,000 and 82,000 complexes are formed after incremental cross-linking of TSH alpha-beta dimer to receptor subunits; and 2) the TSH receptor may be an oligomer composed of three different subunits, 31,000, 17,000, and 63,000, of which only the 31,000 subunit binds TSH.     

Polymorphic glycoprotein-1 on mouse platelets: possible role of Pgp-1 and LFA-1 in antibody-dependent platelet cytotoxicity involving complement

The presence of the Pgp-1 glycoprotein on mouse platelets is demonstrated by antibody-binding techniques, by immunoprecipitation, and by transblotting using the monoclonal antibody (MoAb) C71/26 against Pgp-1. C71/26 immunoprecipitates as a broad band of mol wt 87,000 to 100,000 as determined by radioiodination of the platelet cell surface and by the 3H-sodium borohydride labeling technique. Immunoblotting showed Pgp-1 expression on platelets to be quantitatively similar to its presence on macrophages and resolved platelet Pgp-1 into two bands of mol wt 87,000 and 97,000 whereas Pgp-1 on parasite-elicited peritoneal macrophages showed 82,000 and 87,000 mol wt species. Platelets and monocyte/macrophage cells from either peripheral blood or from the peritoneal cavity showed homogeneous binding of Pgp-1 antibody to greater than 97% of cells by flow cytometry. In contrast, lymphocytes from peripheral blood or from the spleen showed a heterogeneous binding pattern with 20% to 30% of cells being negative, and the majority weakly positive. In functional studies, MoAbs against CR1 and CR3 substantially inhibited platelet immune adherence, whereas C71/26 showed only marginal inhibitor. In contrast, C71/26 and other MoAbs against Pgp-1 inhibited platelet- dependent cytotoxicity of antibody-coated sheep erythrocytes in the presence of C5-deficient mouse plasma whereas M1/70 against CR3 showed no effect. In this assay, MoAbs against the alpha- and beta-subunits of leukocyte functional molecule LFA-1 also inhibited platelet cytotoxicity. These results show that the platelet cell surface moieties Pgp-1 and LFA-1 are involved in or closely associated with antibody-dependent cellular cytotoxicity by platelets.     

A new variant of the alpha subunit of spectrin in hereditary elliptocytosis

A kindred is described in which two brothers with a poikilocytic variant of hereditary elliptocytosis (HE) were found to have a defect of spectrin dimer association and a decreased spectrin-band 3 ratio. Two-dimensional gel electrophoresis of limited tryptic digests of their spectrin revealed decreased amounts of the alpha I domain when compared with control digests and the appearance of two major peptides with mol wts of 43,000 and 42,000 and isoelectric points (5.75 to 5.85) more basic than the alpha I domain. Tryptic digests of spectrin from the asymptomatic mother of the two brothers were normal. Immunoblots of the two-dimensional gels using an antiserum to the alpha I domain revealed that the 43,000- and 42,000-dalton peptides were derived from the alpha I domain, along with a series of lower mol wt peptides, some of which were below the detection limits of Coomassie blue-stained gels. Limit chymotryptic maps of 125I-labeled tryptic peptides confirmed that the 43,000- and 42,000-dalton peptides were derived from the alpha I domain. This kindred represents a new structural variant of spectrin in HE in that the major abnormal tryptic peptides derived from the alpha I domain have lower mol wts and more basic isoelectric points than hitherto described.     

Synthesis and secretion of human chorionic gonadotropin and its subunits in choriocarcinoma cells: a comparative study with normal placental cells.

The human choriocarcinoma cell line, BeWo, synthesizes the glycoprotein hormone, human chorionic gonadotropin (hCG). We have undertaken this study to compare the synthesized and secreted forms of hCG and their alpha- and beta-subunits in cell cultures of BeWo cells to those forms of normal placental cells by immunobinding techniques. BeWo cells appeared to synthesize and secrete one species of the respective hCG subunit. The immature alpha- and beta-subunits, synthesized in BeWo cells as well as those of placental cells, were digested by endoglycosidase H indicating N-linked sugar chain(s) to be the high-mannose type. The mature alpha- and beta-subunits, secreted by BeWo cells as well as subunits of urinary hCG which are usually used as a standard hCG secreted by normal placental cells, were sensitive to neuraminidase treatment indicating that these subunits have terminal sialic acid(s). Contrary to placental cells, mature subunits of BeWo hCG could not be found in any subcellular fraction indicating a rapid secretion rate or supporting the hypothesis that BeWo cells secrete hCG subunits without the formation of secretory granules. The alpha-subunit synthesized in BeWo cells had a slightly lower molecular weight than that of placental cells; however, the alpha-subunit secreted by BeWo cells had a slightly higher molecular weight than the alpha-subunit of urinary hCG. The beta-subunits synthesized and secreted by BeWo cells had slightly higher molecular weights than beta-subunits of both placental cells and urinary hCG. Even after digestion by N-glycanase as well as endoglycosidase H, molecular weights were still different between the respective subunits of BeWo and placental cells indicating that the apoprotein structures of BeWo hCG subunits may differ from those of placental cells. Moreover, urinary beta-subunit was sensitive to endo-alpha-N-acetylgalactosaminidase treatment but the beta-subunit secreted by BeWo cells was not, indicating that the structure of O-linked sugar chain(s), if present, may be unusual. Analysis of assembled and free forms of subunits of BeWo cell cultures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions followed by immunobinding methods revealed that subunits are associated intracellularly and then secreted to the media as hCG. Moreover, only free beta-subunits, but not alpha-subunits, of BeWo hCG were found intra- and extracellularly.     

Calmodulin-binding proteins that interact with actin filaments in a Ca2+-dependent flip-flop manner: survey in brain and secretory tissues.

Regulatory actions of calmodulin on the contractile apparatus and cytoskeleton of smooth muscle and nonmuscle tissue are mediated by a number of specific calmodulin-binding proteins that bind to F-actin in a flip-flop manner--i.e., they bind to calmodulin or F-actin depending on the presence or absence, respectively, of Ca2+. A survey for such proteins in brain, adrenal gland, and pituitary gland identified six polypeptides on polyacrylamide gels--Mr 340,000 (band 1), Mr 240,000/235,000 doublet (band 2), Mr 150,000 (band 3), Mr 129,000 (band 4), Mr 105,000 (band 5), and Mr 94,000 (band 6)--as flip-flop-regulated calmodulin- and F-actin-binding polypeptides. In addition to these polypeptides, a Mr 58,000 non-flip-flop calmodulin-binding actin-binding polypeptide (band 7) was found in all tissues examined. Band 2 was identified as calspectin (spectrin-related protein; fodrin). The flip-flop regulation of calspectin required the presence of a heat-labile nondialyzable factor contained in a supernatant fraction of brain homogenates. Band 1 was distinct from microtubule-associated proteins (MAPs) 1 and 2. However, when band 1 polypeptide was kept on ice 3 days, it converted to a lower molecular weight doublet that migrated with MAP2 on NaDodSO4 gel electrophoresis. Bands 1 and 2 were found in all tissues examined.     

Somatostatin regulation of glycoprotein hormone and free subunit secretion in clinically nonfunctioning and somatotroph adenomas in vitro.

There is increasing evidence that clinically nonfunctioning pituitary tumors produce and secrete glycoprotein hormone and/or free alpha- and beta-subunits. In addition, hypersecretion of free alpha-subunit occurs in up to 37% of patients with somatotroph adenomas. An understanding of glycoprotein hormone regulation is important in developing effective therapeutic strategies for patients with tumors associated with intact glycoprotein hormone and free subunit hypersecretion. We investigated glycoprotein hormone and free subunit secretion by somatostatin in primary dispersed cultures of pituitary tumor cells from 23 patients with pituitary adenomas. Fifteen tumors from patients with clinically nonfunctioning adenomas (group 1) and 8 tumors from patients with somatotroph adenomas and cosecretion of alpha-subunit (group 2) were studied. Cultures were incubated with control or somatostatin-supplemented media for 24 h. Media samples from group 1 tumors were assayed for intact glycoprotein hormones and free alpha- and beta-subunits secretion levels, while media samples from group 2 cultures were assayed for alpha-subunit and GH secretion levels. Significant (P less than 0.05-0.001) inhibition of secretion of 1 or more intact hormones and/or free subunits was found in 10 of the 15 group 1 tumors. SRIF[10(-7) M] suppressed intact gonadotropin secretion in 60% of FSH-producing tumors and 30% of LH-producing tumors. Media concentrations of FSH beta and LH beta were decreased in 31% and 50% of group 1 tumors, respectively, following somatostatin treatment in those tumors which secreted free beta-subunits. alpha-Subunit was secreted by 12 of the 15 tumors, but significant (P less than 0.02-0.01) inhibition by somatostatin was observed in only 2 tumors. In contrast, significant (P less than 0.05-0.001) inhibition of alpha-subunit in the somatotroph adenomas was found in 6 of the 8 tumors. Significant decreases in alpha-subunit were observed only in those tumors where GH was also significantly inhibited by somatostatin. We conclude that 1) somatostatin inhibits intact glycoprotein or free subunit secretion in the majority of clinically nonfunctioning pituitary tumors in vitro and 2) alpha-subunit secretion is suppressed in 17% and 69% of clinically nonfunctioning and somatotroph adenomas, respectively, consistent with a differential regulation of alpha-subunit by somatostatin in these two tumor types.     

Assembly and expression of a synthetic gene encoding the bovine glycoprotein hormone alpha-subunit.

The glycoprotein hormones are a family of alpha beta heterodimeric proteins which are responsible for gonadal and thyroid function. In previous studies we employed chimeric glycoprotein hormone beta-subunits to identify amino acid residues critical for binding to receptors and antibodies. To facilitate similar studies of the alpha-subunit of these hormones, we assembled a 406 bp synthetic gene which encodes the human alpha-subunit leader sequence and the secreted portion of the bovine alpha-subunit. It contains unique restriction sites that can be used for cassette mutagenesis or for making human/bovine alpha-subunit chimeras. The gene was assembled from eight long oligodeoxynucleotides in a single ligation and its structure verified by DNA sequencing. Co-transfection of COS-7 cells with the synthetic gene and the cDNA for human chorionic gonadotropin (hCG) beta-subunit resulted in the secretion of a functional alpha beta heterodimer which bound to luteinizing hormone receptors. The protein was recognized by several monoclonal antibodies including B109, an antibody to a conformational epitope which binds hCG but not the free bovine alpha-, human alpha-, or hCG beta-subunits. This suggests that the binding site for B109 may be formed by residues located primarily within the hCG beta-subunit and that formation of this epitope requires a change in conformation of the beta-subunit when it combines with the alpha-subunit.     

Monoclonal antibodies reveal structural homogeneity of gamma-aminobutyric acid/benzodiazepine receptors in different brain areas.

Monoclonal antibodies (mAb) against a gamma-aminobutyric acid/benzodiazepine receptor complex (GABAA/BZR) were produced by using spleen cells from a mouse immunized with GABAA/BZR purified from bovine cerebral cortex. The mAb, most of which were of the IgG1 isotype could be divided into four groups (I-IV) specifying different antigenic structures. On immunoblots, group I mAb recognized exclusively the Mr 55,000 beta-subunit, while groups II and IV mAb recognized the Mr 50,000 alpha-subunit of bovine GABAA/BZR. Three of the four groups of mAb (I, III, and IV) crossreacted with both human and rat GABAA/BZR with the same subunit specificity as in bovine brain; the fourth group (II) crossreacted with human but not with the rat receptor. The binding sites for benzodiazepines as well as the high and low affinity GABA sites reside on the same structural complex as shown by immunoprecipitation. Ligand binding to these sites was not inhibited by mAb. Since quantitative immunoprecipitation of GABAA/BZR was achieved with mAb selective for either the alpha- or beta-subunit, both subunits occur in each individual receptor complex. The pattern of immunoblot staining suggests that the smaller alpha-subunit is not a processing product of the larger beta-subunit. Both alpha- and beta-subunits were present in all brain areas and species tested (rat cerebral cortex, cerebellum, and hippocampus; bovine cerebral cortex and cerebellum; human cerebral cortex). This suggests a uniform subunit composition of the receptor throughout the brain in contrast to earlier evidence for a heterogeneous subunit composition based on photoaffinity labeling.     

Goblin (ankyrin) in striated muscle: identification of the potential membrane receptor for erythroid spectrin in muscle cells.

Goblin , a high molecular weight (Mr, 260,000) polypeptide of avian erythrocyte plasma membranes characterized by hormone-dependent phosphorylation, is shown by a variety of criteria to be the avian equivalent of ankyrin, the membrane attachment protein for spectrin; a polyclonal monospecific goblin antiserum reacts specifically with ankyrin from mammalian erythrocyte ghosts; goblin and ankyrin have highly homologous, although distinct, two-dimensional peptide maps; and, in reconstitution experiments, goblin binds to spectrin and band 3 in approximately the same molar ratio as ankyrin. Immunoautoradiography and immunofluorescence with goblin antiserum reveal that a serologically related polypeptide (Mr, 235,000) is present in highly purified membrane fractions of mammalian myocardium and in whole extracts of adult chicken cardiac and skeletal muscle-nonerythroid tissues which express predominantly the erythroid (alpha beta-) spectrin phenotype. Erythroid spectrin and goblin (ankyrin) are codistributed in skeletal muscle at the sarcolemma as discrete foci adjacent to the Z lines and, in pectoral muscle, also at the periphery of the Z discs. These spatial relationships indicate that goblin and spectrin in muscle cells form a structural framework that serves as the attachment site for the myofiber at the level of the Z line on the sarcolemma.     

Identification of functional domains of human erythrocyte spectrin.

Isolated human erythrocyte spectrin is a dimer of two unique polypeptide chains. The dimer (alpha beta) undergoes reversible salt- and temperature-dependent association to form (alpha beta)2 tetramers. Spectrin also binds with high affinity to a protein receptor on the cytoplasmic surface of erythrocyte membrane vesicles. By cleavage of spectrin at its cysteine residues with 2-nitro-5-thiocyanobenzoic acid, a 50,000-dalton peptide fragment has been isolated which inhibits the binding of spectrin to erythrocyte membrane vesicles. This peptide arises from a terminal region of the beta chain. An 80,000-dalton peptide generated by restricted trypsin digestion binds preferentially to dimeric spectrin. This peptide arises from a terminal portion of the alpha chain. Multiple peptides involved in noncovalent associations between the chains have also been identified. These associations indicate that the two subunits of spectrin are aligned parallel to one another and that the tetramer formation site and the high-affinity membrane binding site are in close proximity to one another.     

A cell line that produces the glycoprotein hormone alpha-subunit contains specific nuclear factors similar to those present in thyrotropes.

A unique characteristics of thyrotrope-specific gene expression is the coordinated expression and regulation of the alpha- and beta-subunits of TSH. A cell line (alpha TSH) derived from the transplantable mouse thyrotropic tumor MGH101A, which no longer expresses the TSH beta-subunit gene but continues to secrete large amounts of alpha-subunit, was used as a model to study alpha-subunit gene expression independent from the TSH beta-subunit gene and was compared with the expression in TSH-secreting TtT97 tumors. Transient transfection studies showed a striking similarity in the activity of 5' deletions of the mouse alpha-subunit gene promoter in both alpha TSH and TtT97 cells and localized two regions important for expression that spanned 100 base pairs, from -480 to -417 and from -417 to -381. These regions were found to have no activity in nonthyrotrope pituitary GH4 cells and L-cell fibroblasts. Analysis of the alpha-subunit 5' flanking DNA interactions with alpha TSH and TtT97 nuclear extracts showed two DNase I protected sequences, from -474 to -452 and from -447 to -400, both of which colocalized with the functionally important regions. Gel retardation analysis demonstrated the specificity of these interactions, and a similar migration of the DNA-protein complexes suggested that protein factors were similar in the two cell types. We conclude that the nuclear factors necessary for alpha-subunit expression in thyrotropes are retained in alpha TSH cells. Moreover, since alpha TSH cells do not express the TSH beta-subunit gene, the factors that determine the expression of the alpha-subunit may not be sufficient for TSH beta-subunit gene expression.     

Immunochemical delineation of an oncofetal antigen on normal and simian virus 40-transformed human fetal melanocytes.

Human melanoma cells of uveal origin shed 94,000- and 240,000-dalton glycoproteins in common with most melanoma cell lines of dermal origin. Normal human melanocytes derived from fetal uvea shed a 90,000-dalton glycoprotein that was found to be immunologically identical with the 94,000-dalton glycoprotein of melanoma cells. Expression of this 90,000-dalton molecule was confined to fetal cells of ectodermal origin. After simian virus 40 (SV40) transformation of human fetal melanocytes, there was an apparent increase in molecular size of this component to 94,000 daltons. In contrast, the 240,000-dalton glycoprotein was not synthesized or shed by uninfected or SV40-transformed fetal melanocytes. These data suggest that the 94,000-dalton glycoprotein is an oncofetal antigen of ectodermal origin.     

Senescent cell antigen is immunologically related to band 3.

IgG autoantibodies in human serum selectively bind to a glycopeptide antigen that appears on senescent and damaged cells in situ. We identified the membrane protein from which the senescent cell antigen is derived by using a phagocytosis-inhibition assay and immunoautoradiographic gel staining and electroblotting techniques. Results of the phagocytosis-inhibition assay revealed that only the purified transmembrane glycoprotein designated "band 3" and senescent cell antigen inhibited the phagocytosis of erythrocytes induced by IgG eluted from senescent erythrocytes. Purified spectrin, syndein, band 4.1, actin, glycophorin A, and intact or desialylated sialoglycoprotein periodic acid/Schiff (PAS) staining bands 1-4 containing glycophorins A, B, and C did not inhibit phagocytosis. Specific antibodies against the senescent cell antigen and erythrocyte band 3 were used to identify the membrane protein from which the senescent cell antigen is derived. Band 3-related polypeptides (MrS approximately equal to 60,000, 42,000, and 18-26,000) were identified in erythrocyte ghosts prepared in the presence of diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, and EDTA by immunoautoradiography with antiband 3. Antibodies to senescent cell antigen reacted with band 3 and the same lower Mr band 3-related polypeptides. Thus, the senescent cell antigen is immunologically related to band 3.