scholarly journals A human leukocyte differentiation antigen family with distinct alpha-subunits and a common beta-subunit: the lymphocyte function-associated antigen (LFA-1), the C3bi complement receptor (OKM1/Mac-1), and the p150,95 molecule.

1983 ◽  
Vol 158 (6) ◽  
pp. 1785-1803 ◽  
Author(s):  
F Sanchez-Madrid ◽  
J A Nagy ◽  
E Robbins ◽  
P Simon ◽  
T A Springer
Keyword(s):  
Human Leukocyte ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Complement Receptor ◽  
Lymphocyte Function ◽  
Subunit Dissociation ◽  
Molecular Weights ◽  
Alpha Subunits ◽  
Sds Page

The human lymphocyte function-associated antigen-1 (LFA-1), the complement receptor-associated OKM1 molecule, and a previously undescribed molecule termed p150,95, have been found to be structurally and antigenically related. Each antigen contains an alpha- and beta-subunit noncovalently associated in an alpha 1 beta 1-structure as shown by cross-linking experiments. LFA-1, OKM1, and p150,95 alpha-subunit designations and their molecular weights are alpha L = 177,000 Mr, alpha M = 165,000 Mr, and alpha X = 150,000 Mr, respectively. The beta-subunits are all = 95,000 Mr. Some MAb precipitated only LFA-1, others only OKM1, and another precipitates all three antigens. The specificity of these MAb for particular subunits was examined after subunit dissociation by high pH. MAb specific for LFA-1 or OKM1 bind to the alpha L- or alpha M-subunits, respectively, while the cross-reactive MAb binds to the beta-subunits. Coprecipitation experiments with intact alpha 1 beta 1-complexes showed anti-alpha and anti-beta MAb can precipitate the same molecules. In two-dimensional (2D) isoelectric focusing-SDS-PAGE, the alpha subunits of the three antigens are distinct, while the beta-subunits are identical. Biosynthesis experiments showed alpha L, alpha M, and alpha X are synthesized from distinct precursors, as is beta. The three antigens differ in expression on lymphocytes, granulocytes, and monocytes. During maturation of the monoblast-like U937 line, alpha M and alpha X are upregulated and alpha L is downregulated. Some MAb to the alpha subunit of OKM1 inhibited the complement receptor type three. LFA-1, OKM1, and p150,95 constitute a novel family of functionally important human leukocyte antigens that share a common beta-subunit.

scholarly journals Mapping of antigenic and functional epitopes on the alpha- and beta-subunits of two related mouse glycoproteins involved in cell interactions, LFA-1 and Mac-1.

1983 ◽  
Vol 158 (2) ◽  
pp. 586-602 ◽  
Author(s):  
F Sanchez-Madrid ◽  
P Simon ◽  
S Thompson ◽  
T A Springer
Keyword(s):  
Monoclonal Antibodies ◽  
Structural Features ◽  
Cell Interactions ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Complement Receptor ◽  
Receptor Function ◽  
Alpha Subunits ◽  
Sds Page

Mouse Mac-1, a complement receptor-associated surface structure on macrophages, and LFA-1, a function-associated structure on lymphocytes, comprise a novel family of leukocyte differentiation antigens participating in adhesive cell interactions. Mac-1 and LFA-1 contain alpha-subunits of 170,000 and 180,000 Mr, respectively, and beta-subunits of 95,000 Mr noncovalently associated in alpha 1 beta 1 complexes. The structural relation between the alpha- and between the beta-subunits, and the location of functionally important sites on the molecules, have been probed with antibodies. Both non-cross-reactive and cross-reactive monoclonal antibodies (MAb) and antisera prepared to the purified molecules or the LFA-1 alpha-subunits were used. Reactivity with individual subunits was studied by immunoprecipitation after dissociation induced by high pH treatment, or by immunoblotting after SDS-PAGE. Cross-reactive epitopes on Mac-1 and LFA-1 were found to be present on the beta-subunits, which were immunologically identical. Non-cross-reactive epitopes that are distinctive for Mac-1 or LFA-1 were localized to the alpha-subunits. MAb to LFA-1 alpha-subunit epitopes inhibited CTL-mediated killing. Two MAb to Mac-1 alpha-subunit epitopes but not a third MAb to a spatially distinct alpha-epitope inhibited complement receptor function. Neither function was inhibited by a MAb binding to a common beta-subunit epitope. Therefore, sites of Mac-1 and LFA-1 involved in their respective adhesion-related functions, as well as distinctive structural features, have been localized to the alpha-subunits.

Analysis of subunit assembly of the Na-K-ATPase

1994 ◽  
Vol 266 (3) ◽  
pp. C579-C589 ◽  
Author(s):  
D. M. Fambrough ◽  
M. V. Lemas ◽  
M. Hamrick ◽  
M. Emerick ◽  
K. J. Renaud ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Sodium Pump ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Limited Region ◽  
Subunit Assembly ◽  
Alpha Subunits ◽  
Subunit Isoforms ◽  
P Type

The Na-K-ATPase, or sodium pump, is comprised of two subunits, alpha and beta. Each subunit spans the lipid bilayer of the cell membrane. This review summarizes our efforts to determine how the two subunits interact to form the functional ion transporter. Our major approach has been to observe the potential for subunit assembly when one or both subunits are truncated or present as chimeras that retain only a limited region of the Na-K-ATPase. DNAs encoding these altered subunit forms of the avian Na-K-ATPase are expressed in mammalian cells. Monoclonal antibodies specific for the avian beta-subunit are then used to purify newly synthesized avian beta-subunits, and the presence of accompanying alpha-subunits indicates that subunit assembly has occurred. The ectodomain of the beta-subunit (approximately residues 62-304) is sufficient for assembly with the alpha-subunit, and a COOH-terminal truncation of the beta-subunit that lacks aminoacyl residues beyond 162 will assemble inefficiently. A maximum of 26 aminoacyl residues of the alpha-subunit are necessary for robust assembly with the beta-subunit, when this sequence replaces the COOH-terminal half of the loop between membrane spans 7 and 8 in the SERCA1 Ca-ATPase. This region of the Ca-ATPase faces the lumen of the endoplasmic reticulum. These findings encourage study of other related questions, including whether there is preferential assembly of certain subunit isoforms and how various P-type ATPases are targeted to their appropriate subcellular compartments.

scholarly journals Protein kinase activity of the insulin receptor

1986 ◽  
Vol 235 (1) ◽  
pp. 1-11 ◽  
Author(s):  
S Gammeltoft ◽  
E Van Obberghen
Keyword(s):  
Insulin Receptor ◽  
Molecular Mechanisms ◽  
Kinase Activity ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Protein Kinase Activity ◽  
Intact Cells ◽  
Receptor Complexes ◽  
Alpha Subunits

The insulin receptor is an integral membrane glycoprotein (Mr approximately 300,000) composed of two alpha-subunits (Mr approximately 130,000) and two beta-subunits (Mr approximately 95,000) linked by disulphide bonds. This oligomeric structure divides the receptor into two functional domains such that alpha-subunits bind insulin and beta-subunits possess tyrosine kinase activity. The amino acid sequence deduced from cDNA of the single polypeptide chain precursor of human placental insulin receptor revealed that alpha- and beta-subunits consist of 735 and 620 residues, respectively. The alpha-subunit is hydrophilic, disulphide-bonded, glycosylated and probably extracellular. The beta-subunit consists of a short extracellular region which links the alpha-subunit through disulphide bridges, a hydrophobic transmembrane region and a longer cytoplasmic region which is structurally homologous with other tyrosine kinases like the src oncogene product and EGF receptor kinases. The cellular function of insulin receptors is dual: transmembrane signalling and endocytosis of hormone. The binding of insulin to its receptor on the cell membrane induces transfer of signal from extracellular to cytoplasmic receptor domains leading to activation of cell metabolism and growth. In addition, hormone-receptor complexes are internalized leading to intracellular proteolysis of insulin, whereas receptors are recycled to the membrane. These phenomena are kinetically well-characterized, but their molecular mechanisms remain obscure. Insulin receptor in different tissues and animal species are homologous in their structure and function, but show also significant differences regarding size of alpha-subunits, binding kinetics, insulin specificity and receptor-mediated degradation. We suggest that this heterogeneity of receptors may be linked to the diversity in insulin effects on metabolism and growth in various cell types. The purified insulin receptor phosphorylates its own beta-subunit and exogenous protein and peptide substrates on tyrosine residues, a reaction which is insulin-sensitive, Mn2+-dependent and specific for ATP. Tyrosine phosphorylation of the beta-subunit activates receptor kinase activity, and dephosphorylation with alkaline phosphatase deactivates the kinase. In intact cells or impure receptor preparations, a serine kinase is also activated by insulin. The cellular role of two kinase activities associated with the insulin receptor is not known, but we propose that the tyrosine- and serine-specific kinases mediate insulin actions on metabolism and growth either through dual-signalling or sequential pathways.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Characterization of beta pat-3 heterodimers, a family of essential integrin receptors in C. elegans.

1995 ◽  
Vol 129 (4) ◽  
pp. 1127-1141 ◽  
Author(s):  
S N Gettner ◽  
C Kenyon ◽  
L F Reichardt
Keyword(s):  
Caenorhabditis Elegans ◽  
Cytoplasmic Domain ◽  
Beta Subunit ◽  
Beta Subunits ◽  
Integrin Receptors ◽  
Diverse Range ◽  
C Elegans ◽  
Alpha Subunits ◽  
Sds Page

Members of the integrin family of cell surface receptors have been shown to mediate a diverse range of cellular functions that require cell-cell or cell-extracellular matrix interactions. We have initiated the characterization of integrin receptors from the nematode Caenorhabditis elegans, an organism in which genetics can be used to study integrin function with single cell resolution. Here we report the cloning of an integrin beta subunit from C. elegans which is shown to rescue the embryonic lethal mutation pat-3(rh54) and is thus named beta pat-3. Analysis of the deduced amino acid sequence revealed that beta pat-3 is more similar to Drosophila integrin beta PS and to vertebrate integrin beta 1 than to other integrin beta subunits. Regions of highest homology are in the RGD-binding region and in the cytoplasmic domain. In addition, the 56 cysteines present in the majority of integrin beta subunits are conserved. A major transcript of approximately 3 kilo-base pairs was detected by RNA blot analysis. Immunoblot analysis using a polyclonal antiserum against the cytoplasmic domain showed that beta pat-3 migrates in SDS-PAGE with apparent M(r) of 109 k and 120 k under nonreducing and reducing conditions, respectively. At least nine protein bands with relative molecular weights in the range observed for known integrin alpha subunits coprecipitate with beta pat-3, and at least three of these bands migrate in SDS-PAGE with increased mobility when reduced. This behavior has been observed for a majority of integrin alpha subunits. Immunoprecipitations of beta pat-3 from developmentally staged populations of C. elegans showed that the expression of several of these bands changes during development. The monoclonal antibody MH25, which has been postulated to recognize the transmembrane component of the muscle dense body structure a (Francis, G. R., and R. H. Waterston. 1985. Muscle organization in Caenorhabditis elegans: localization of proteins implicated in thin filament attachment and I-band organization. J. Cell Biol. 101:1532-1549), was shown to recognize beta pat-3. Finally, immunocytochemical analysis revealed that beta pat-3 is expressed in the embryo and in many cell types postembryonically, including muscle, somatic gonad, and coelomocytes, suggesting multiple roles for integrin heterodimers containing this beta subunit in the developing animal.

scholarly journals Investigations into the post-translational modification and mechanism of isopenicillin N:acyl-CoA acyltransferase using electrospray mass spectrometry

1993 ◽  
Vol 294 (2) ◽  
pp. 357-363 ◽  
Author(s):  
R T Aplin ◽  
J E Baldwin ◽  
P L Roach ◽  
C V Robinson ◽  
C J Schofield
Keyword(s):  
Mass Spectrometry ◽  
Electrospray Mass Spectrometry ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
British Library ◽  
Beta Subunits ◽  
Post Translational Modification ◽  
Acetic Acids

Electrospray mass spectrometry (e.s.m.s.) was used to confirm the position of the post-translational cleavage of the isopenicillin N:acyl-CoA acyltransferase preprotein to give the alpha- and beta-subunits. The e.s.m.s. studies suggested partial modification of the alpha-subunit in vivo by exogenously added substituted acetic acids. E.s.m.s. has also allowed the observation in vitro of the transfer of the acyl group from several acyl-CoAs to the beta-subunit. N.m.r. data for the CoA species have been deposited as Supplementary Publication SUP 500173 (2 pages) at the British Library Document Supply Centre (DSC), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, from whom copies can be obtained on the terms indicated in Biochem. J. (1993) 289, 9.

scholarly journals Molecular-mass heterogeneity of Griffonia simplicifolia lectin IV subunits. Differences in the oligosaccharide moieties in the N-terminal region

1990 ◽  
Vol 272 (2) ◽  
pp. 343-350 ◽  
Author(s):  
P V Nikrad ◽  
J R Pearlstone ◽  
M R Carpenter ◽  
R U Lemieux ◽  
L B Smillie
Keyword(s):  
Monosaccharide Composition ◽  
Unique Sequence ◽  
Beta Subunit ◽  
The Other ◽  
Alpha Subunit ◽  
Sequence Analyses ◽  
Griffonia Simplicifolia ◽  
Sds Page ◽  
Dimeric Protein ◽  
Covalently Linked

Lectin IV of Griffonia simplicifolia (Mr approximately 56,000), which has a strong affinity for both the Lewis b and Y blood-group determinants, is a dimeric protein of two subunits, alpha (29 kDa) and beta (27 kDa), separable by SDS/PAGE and containing covalently linked oligosaccharide. After digestion with N-glycanase, the protein migrates as a single band with a mobility identical with that of the beta-subunit. After cleavage with hydroxylamine of 3H-labelled, but otherwise intact, lectin, the radioactively labelled oligosaccharide was found to be associated with two blocked N-terminal peptides separable by h.p.l.c. and having identical amino acid compositions. One of these had three or four glucosamine residues per molecule, whereas the other had only one or two. Sequence analyses of these, as well as of a 21 kDa hydroxylamine-cleaved fragment and of the intact lectin pretreated with pyroglutamate aminopeptidase, have provided a unique sequence for residues 1-62 of the two subunits. Evidence is presented for two sites of N-linked oligosaccharide attachment at Asn-5 and Asn-18. Whereas the alpha-subunit has oligosaccharide linked to both sites, the beta-subunit has carbohydrate associated with only one (Asn-18). Sugar analyses of the whole lectin reveal a monosaccharide composition of (Xyl)3(Fuc)2(Man)10(GlcNAc)6, representing 6.4% of the mass of the molecule. Taken together with the susceptibility of the Asn-5 linkage (but not of Asn-18) to N-glycanase digestion, the observations indicate that the structures of the oligosaccharides at residues 5 and 18 are different.

scholarly journals Functional reconstitution of the human interleukin-3 receptor

Blood ◽  
1992 ◽  
Vol 80 (1) ◽  
pp. 84-90 ◽  
Author(s):  
T Kitamura ◽  
A Miyajima
Keyword(s):  
Ligand Binding ◽  
Growth Response ◽  
Human Factor ◽  
Beta Subunit ◽  
Beta Subunits ◽  
Stable Transfectants ◽  
Interleukin 3 ◽  
Gm Csf ◽  
High Affinity ◽  
Alpha Subunits

The high-affinity receptors for human interleukin-3 (IL-3), GM-CSF, and IL-5 are composed of alpha and beta subunits. The alpha subunits are primary ligand binding proteins specific for each ligand, whereas the three human receptors share a common beta subunit (beta c). In contrast to humans mice have two closely related genes, AIC2A and AIC2B, which are homologous to human beta c. The AIC2A gene encodes a low-affinity murine IL-3 binding protein, and the AIC2B protein is the beta subunit shared between murine GM-CSF receptors (mGMR) and IL-5 receptors (mIL- 5R). To examine the function of these receptor components, we established various stable transfectants of murine IL-2-dependent CTLL- 2 cells. CTLL-2 transfectants expressing both the alpha and beta subunits of the human IL-3 receptor (hIL-3R) proliferated in response to physiologic concentrations of hIL-3. Coexpression of hIL-3R alpha with AIC2B but not with AIC2A in CTLL-2 cells conferred a growth response to hIL-3. Although CTLL-2 transfectants expressing hIL-3R alpha alone did not proliferate in the presence of hIL-3, hIL-3- responsive sublines were repeatedly isolated. These sublines expressed endogenous AIC2B but not AIC2A. These results indicate that human beta c is essential for hIL-3 signaling and that AIC2B is a murine equivalent of human beta c. We also showed that hIL-3 and hGM-CSF induced tyrosine phosphorylation of several proteins in CTLL transfectants, similar to those observed in human factor-dependent TF-1 cells stimulated with hIL-3 and hGM-CSF.

scholarly journals Functional reconstitution of the human interleukin-3 receptor

Blood ◽  
1992 ◽  
Vol 80 (1) ◽  
pp. 84-90 ◽  
Author(s):  
T Kitamura ◽  
A Miyajima
Keyword(s):  
Ligand Binding ◽  
Growth Response ◽  
Human Factor ◽  
Beta Subunit ◽  
Beta Subunits ◽  
Stable Transfectants ◽  
Interleukin 3 ◽  
Gm Csf ◽  
High Affinity ◽  
Alpha Subunits

Abstract The high-affinity receptors for human interleukin-3 (IL-3), GM-CSF, and IL-5 are composed of alpha and beta subunits. The alpha subunits are primary ligand binding proteins specific for each ligand, whereas the three human receptors share a common beta subunit (beta c). In contrast to humans mice have two closely related genes, AIC2A and AIC2B, which are homologous to human beta c. The AIC2A gene encodes a low-affinity murine IL-3 binding protein, and the AIC2B protein is the beta subunit shared between murine GM-CSF receptors (mGMR) and IL-5 receptors (mIL- 5R). To examine the function of these receptor components, we established various stable transfectants of murine IL-2-dependent CTLL- 2 cells. CTLL-2 transfectants expressing both the alpha and beta subunits of the human IL-3 receptor (hIL-3R) proliferated in response to physiologic concentrations of hIL-3. Coexpression of hIL-3R alpha with AIC2B but not with AIC2A in CTLL-2 cells conferred a growth response to hIL-3. Although CTLL-2 transfectants expressing hIL-3R alpha alone did not proliferate in the presence of hIL-3, hIL-3- responsive sublines were repeatedly isolated. These sublines expressed endogenous AIC2B but not AIC2A. These results indicate that human beta c is essential for hIL-3 signaling and that AIC2B is a murine equivalent of human beta c. We also showed that hIL-3 and hGM-CSF induced tyrosine phosphorylation of several proteins in CTLL transfectants, similar to those observed in human factor-dependent TF-1 cells stimulated with hIL-3 and hGM-CSF.

scholarly journals Genetic identification of residues involved in association of alpha and beta G-protein subunits.

1994 ◽  
Vol 14 (5) ◽  
pp. 3223-3229 ◽  
Author(s):  
M Whiteway ◽  
K L Clark ◽  
E Leberer ◽  
D Dignard ◽  
D Y Thomas
Keyword(s):  
G Protein ◽  
Protein A ◽  
Beta Subunit ◽  
Yeast Cells ◽  
Genetic Identification ◽  
Alpha Subunit ◽  
Protein Subunit ◽  
Direct Role ◽  
Subunit Dissociation ◽  
Allele Specific

The GPA1, STE4, and STE18 genes of Saccharomyces cerevisiae encode the alpha, beta, and gamma subunits, respectively, of a G protein involved in the mating response pathway. We have found that mutations G124D, W136G, W136R, and delta L138 and double mutations W136R L138F and W136G S151C of the Ste4 protein cause constitutive activation of the signaling pathway. The W136R L138F and W136G S151C mutant Ste4 proteins were tested in the two-hybrid protein association assay and found to be defective in association with the Gpa1 protein. A mutation at position E307 of the Gpa1 protein both suppresses the constitutive signaling phenotype of some mutant Ste4 proteins and allows the mutant alpha subunit to physically associate with a specific mutant G beta subunit. The mutation in the Gpa1 protein is adjacent to the hinge, or switch, region that is required for the conformational change which triggers subunit dissociation, but the mutation does not affect the interaction of the alpha subunit with the wild-type beta subunit. Yeast cells constructed to contain only the mutant alpha and beta subunits mate and respond to pheromones, although they exhibit partial induction of the pheromone response pathway. Because the ability of the modified G alpha subunit to suppress the Ste4 mutations is allele specific, it is likely that the residues defined by this analysis play a direct role in G-protein subunit association.

scholarly journals LFA-1 immunodeficiency disease. Definition of the genetic defect and chromosomal mapping of alpha and beta subunits of the lymphocyte function-associated antigen 1 (LFA-1) by complementation in hybrid cells.

1986 ◽  
Vol 164 (3) ◽  
pp. 855-867 ◽  
Author(s):  
S D Marlin ◽  
C C Morton ◽  
D C Anderson ◽  
T A Springer
Keyword(s):  
Genetic Defect ◽  
Surface Expression ◽  
Beta Subunit ◽  
Chromosome 21 ◽  
Chromosomal Mapping ◽  
Cell Surface Expression ◽  
Beta Subunits ◽  
Lymphocyte Function ◽  
Disease Definition ◽  
Immunodeficiency Disease

Lymphocyte function associated antigen 1 (LFA-1) is a leukocyte cell adhesion protein. We have studied a novel human immunodeficiency disease in which LFA-1 and two other proteins which share the same beta subunit are lacking from the surface of leukocytes. The basis of the inherited defect in cell surface expression of both the alpha and beta subunits of LFA-1 was determined by somatic cell fusion of patient or normal human cells with an LFA-1+ mouse T cell line. Human LFA-1 alpha and beta subunits from normal cells could associate with mouse LFA-1 subunits to form interspecies hybrid alpha beta complexes. Surface expression of the alpha but not the beta subunit of patient cells was rescued by the formation of interspecies complexes. The findings show that the LFA-1 alpha subunit in genetically deficient cells is competent for surface expression in the presence of an appropriate beta subunit, and suggest that the genetic lesion affects the beta subunit. The human LFA-1 alpha and beta subunits were mapped to chromosomes 16 and 21, respectively. The genetic defect is inferred to be on chromosome 21.

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