Primary structure of the leukocyte function-associated molecule-1 alpha subunit: an integrin with an embedded domain defining a protein superfamily.
ABSTRACT: The leukocyte function-associated molecule 1 (LFA-1, CD11a/CD18) is a membrane glycoprotein which functions in cell-cell adhesion by heterophilic interaction with intercellular adhesion molecule 1 (ICAM-1). LFA-1 consists of an alpha subunit (Mr = 180,000) and a beta subunit (Mr = 95,000). We report the molecular biology and protein sequence of the alpha subunit. Overlapping cDNAs containing 5,139 nucleotides were isolated using an oligonucleotide specified by tryptic peptide sequence. The mRNA of 5.5 kb is expressed in lymphoid and myeloid cells but not in a bladder carcinoma cell line. The protein has a 1,063-amino acid extracellular domain, a 29-amino acid transmembrane region, and a 53-amino acid cytoplasmic tail. The extracellular domain contains seven repeats. Repeats V-VII are in tandem and contain putative divalent cation binding sites. LFA-1 has significant homology to the members of the integrin superfamily, having 36% identity with the Mac-1 and p150,95 alpha subunits and 28% identity with other integrin alpha subunits. An insertion of approximately 200 amino acids is present in the NH2-terminal region of LFA-1. This "inserted/interactive" or I domain is also present in the p150,95 and Mac-1 alpha subunits but is absent from other integrin alpha subunits sequenced to date. The I domain has striking homology to three repeats in human von Willebrand factor, two repeats in chicken cartilage matrix protein, and a region of complement factor B. These structural features indicate a bipartite evolution from the integrin family and from an I domain family. These features may also correspond to relevant functional domains.
Project description:Integrin heterodimers mediate a variety of adhesive interactions, including neuronal attachment to and process outgrowth on laminin. We report here the cloning and primary sequence of an M-200 kD integrin alpha subunit that associates with the integrin beta 1 subunit to form a receptor for both laminin and collagen. Similarities in ligand-binding specificity, relative molecular mass and NH2-terminal sequence make this a strong candidate for the rat homologue of the alpha subunit of the human integrin VLA-1. The full-length rat alpha 1 cDNAs encode a protein containing a purative signal sequence and a mature polypeptide of 1,152 amino acids, with extracellular, transmembrane and cytoplasmic domains. Several structural features are conserved with other integrin alpha chains, including (a) a sequence motif repeated seven times in the NH2-terminal half; (b) potential Ca2+/Mg2+ binding sites in repeats 5, 6, and 7, and (c) alignment of at least 14 of 23 cysteine residues. This rat alpha 1 sequence also contains a 206-amino acid I domain, inserted between repeats 2 and 3, that is homologous to I domains found in the same position in the alpha subunits of several integrins (VLA-2, Mac-1, LFA-1, p150). The rat alpha 1 and human VLA-2 apha subunits share greater than 50% sequence identity in the seven repeats and I domain, suggesting that these sequence identities may underlie some of their similar ligand-binding specificities. However, the rat integrin alpha 1 subunit has several unique features, including a 38-residue insert between two Ca2+/Mg2+ binding domains, and a divergent 15-residue cytoplasmic sequence, that may potentially account for unique functions of this integrin.
Project description:The receptor on human neutrophils (polymorphonuclear leukocytes) that mediates cellular adherence consists of two noncovalently associated subunits, designated alpha M (Mac-1 alpha, Mol alpha, or CD11b; Mr, 170,000) and beta (Mac-1 beta, Mol beta, or CD18; Mr, 100,000). We isolated a cDNA clone for the human neutrophil alpha M subunit by screening a lambda gt 11 cDNA library made from chronic myelogenous leukemia neutrophils by using an affinity-purified rabbit polyclonal antibody directed against the alpha M subunit. We used this cDNA clone to obtain additional clones from cDNA libraries made from differentiated HL-60 promyelocytic leukemia cells. Together these cDNAs constitute the complete 1137-amino acid sequence for the mature human alpha M subunit protein. The deduced amino acid sequence indicates the presence of an extensive extracellular domain with three putative metal-binding regions, (i) an amino acid region that is homologous to the A domain of von Willebrand factor, (ii) a 26-amino acid hydrophobic sequence that is a potential transmembrane domain, and (iii) a 19-amino acid cytoplasmic region. The amino acid sequence for the human neutrophil alpha M subunit contains regions that are closely related to amino acid sequences of adhesion receptors belonging to the integrin family.
Project description:?? integrin of lymphocyte function-associated antigen-1 (LFA-1) or macrophage-1 antigen (Mac-1) binds to their common ligand of intercellular adhesion molecule-1 (ICAM-1) and mediates leukocyte-endothelial cell (EC) adhesions in inflammation cascade. Although the two integrins are known to have distinct functions, the corresponding micro-structural bases remain unclear. Here (steered-)molecular dynamics simulations were employed to elucidate the conformational stability of ? subunit I domains of LFA-1 and Mac-1 in different affinity states and relevant I domain-ICAM-1 interaction features. Compared with low affinity (LA) Mac-1, the LA LFA-1 I domain was unstable in the presence or absence of ICAM-1 ligand, stemming from diverse orientations of its ??-helix with different motifs of zipper-like hydrophobic junction between ??- and ??-helices. Meanwhile, spontaneous transition of LFA-1 I domain from LA state to intermediate affinity (IA) state was first visualized. All the LA, IA, and high affinity (HA) states of LFA-1 I domain and HA Mac-1 I domain were able to bind to ICAM-1 ligand effectively, while LA Mac-1 I domain was unfavorable for binding ligand presumably due to the specific orientation of S144 side-chain that capped the MIDAS ion. These results furthered our understanding in correlating the structural bases with their functions of LFA-1 and Mac-1 integrins from the viewpoint of I domain conformational stability and of the characteristics of I domain-ICAM-1 interactions.
Project description:cDNAs for another beta subunit of the integrin family were isolated with the aid of polymerase chain reaction and sequenced. The combined cDNA sequence is 3110 base pairs (bp) in size and has one long open reading frame of 2388 bp. The deduced amino acid sequence is similar to those of other integrin beta subunits but does not correspond to beta 1, beta 2, beta 3, or beta 4 subunits. This beta subunit is divided by a membrane-spanning domain into a large extracellular domain at the N-terminal side and a small intracellular domain at the C-terminal side. The extracellular domain has a cysteine-rich region that contains four repeats of 8-cysteine motifs. All 56 cysteine residues found in the extracellular domains of other mature beta subunits are present in this beta subunit. The beta subunit reported here has particularly high homology with the beta 3 subunit. The mRNA for the molecule is approximately 3.5 kbp in size and is expressed in various cell types. Other researchers have recently reported additional beta subunits that associate with the vitronectin receptor alpha subunit. The deduced amino acid sequence of this molecule contains the N-terminal partial amino acid sequence of one of these beta subunits, beta x. The beta subunit described herein seems to be identical to the beta x subunit and to function as the beta subunit of a vitronectin receptor.
Project description:The N-terminal approximately 440 aa of integrin alpha subunits contain seven sequence repeats. These are predicted here to fold into a beta-propeller domain. A homologous domain from the enzyme phosphatidylinositol phospholipase D is predicted to have the same fold. The domains contain seven four-stranded beta-sheets arranged in a torus around a pseudosymmetry axis. The trimeric G-protein beta subunit (G beta) appears to be the most closely related beta-propeller. Integrin ligands and a putative Mg2+ ion are predicted to bind to the upper face of the beta-propeller. This face binds substrates in beta-propeller enzymes and is used by the G protein beta subunit to bind the G protein alpha subunit. The integrin alpha subunit I domain, which is structurally homologous to the G protein alpha subunit, is tethered to the top of the beta-propeller domain by a hinge that may allow movement of the domains relative to one another. The Ca2+-binding motifs in integrin alpha subunits are on the lower face of the beta-propeller.
Project description:Repeats-in-toxin leukotoxin (LtxA) produced by the oral bacterium Aggregatibacter actinomycetemcomitans kills human leukocytes in a lymphocyte function-associated antigen 1 (LFA-1, integrin ?L /?2 )-dependent manner, although the mechanism for this interaction has not been identified. The LtxA internalisation by LFA-1-expressing cells was explored with florescence resonance energy transfer (FRET) microscopy using a cell line that expresses LFA-1 with a cyan fluorescent protein-tagged cytosolic ?L domain and a yellow fluorescent protein-tagged ?2 domain. Phorbol 12-myristate 13-acetate activation of LFA-1 caused transient cytosolic domain separation. However, addition of LtxA resulted in an increase in FRET, indicating that LtxA brings the cytosolic domains closer together, compared with the inactive state. Unlike activation, this effect was not transient, lasting more than 30 min. Equilibrium constants of LtxA binding to the cytoplasmic domains of both ?L and ?2 were determined using surface plasmon resonance. LtxA has a strong affinity for the cytosolic domains of both the ?L and ?2 subunits (Kd = 15 and 4.2 nM, respectively) and a significantly lower affinity for the cytoplasmic domains of other integrin ?M , ?X , and ?3 subunits (Kd = 400, 180, and 230 nM, respectively), used as controls. Peptide fragments of ?L and ?2 show that LtxA binds membrane-proximal domain of ?L and intermediate domain of ?2 .
Project description:BACKGROUND: Lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18, alphaLbeta2), the most abundant and widely expressed beta2-integrin, is required for many cellular adhesive interactions during the immune response. Many studies have shown that LFA-1 is centrally involved in the pathogenesis of several diseases caused by Repeats-in-toxin (RTX)-producing bacteria. RESULTS: The porcine-LFA-1 CD11a (alpha) subunit coding sequence was cloned, sequenced and compared with the available mammalian homologues in this study. Despite some focal differences, it shares all the main characteristics of these latter. Interestingly, as in sheep and humans, an allelic variant with a triplet insertion resulting in an additional Gln-744 was consistently identified, which suggests an allelic polymorphism that might be biologically relevant. CONCLUSION: Together with the pig CD18-encoding cDNA, which has been available for a long time, the sequence data provided here will allow the successful expression of porcine CD11a, thus giving the first opportunity to express the Sus scrofa beta2-integrin LFA-1 in vitro as a tool to examine the specificities of inflammation in the porcine species.
Project description:During inflammation, circulating polymorphonuclear neutrophils (PMNs) receive signals to cross the endothelial barrier and migrate through the extracellular matrix (ECM) to reach the injured site. Migration requires complex and poorly understood interactions of chemokines, chemokine receptors, ECM molecules, integrins, and other receptors. Here we show that the ECM protein lumican regulates PMN migration through interactions with specific integrin receptors. Lumican-deficient (Lum(-/-)) mice manifest connective tissue defects, impaired innate immune response, and poor wound healing with reduced PMN infiltration. Lum(-/-) PMNs exhibit poor chemotactic migration that is restored with exogenous recombinant lumican and inhibited by anti-lumican antibody, confirming a role for lumican in PMN migration. Treatment of PMNs with antibodies that block beta(2), beta(1), and alpha(M) integrin subunits inhibits lumican-mediated migration. Furthermore, immunohistochemical and biochemical approaches indicate binding of lumican to beta(2), alpha(M), and alpha(L) integrin subunits. Thus, lumican may regulate PMN migration mediated by MAC-1 (alpha(M)/beta(2)) and LFA-1 (alpha(L)/beta(2)), the two major PMN surface integrins. We detected lumican on the surface of peritoneal PMNs and not bone marrow or peripheral blood PMNs. This suggests that PMNs must acquire lumican during or after crossing the endothelial barrier as they exit circulation. We also found that peritoneal PMNs do not express lumican, whereas endothelial cells do. Taken together these observations suggest a novel endothelial lumican-mediated paracrine regulation of neutrophils early on in their migration path.
Project description:The complete amino acid sequence of the L-type calcium channel alpha 1 subunit from the carp (Cyprinus carpio) white skeletal muscle was deduced by cDNA cloning and sequence analysis. The open reading frame encodes 1852 amino acids (Mr 210,060). A 155-amino acid COOH-terminal sequence (after the fourth internal repeat) is evolutionarily preserved (90% homology) and may represent an important functional domain of L-type calcium channels. The photolabeled, membrane-bound, and purified carp alpha 1 subunits have masses of 211 and 190 kDa. The purified channel could not be phosphorylated by cAMP-dependent protein kinase. Two glycoproteins (alpha 2 subunits) are associated with the alpha 1 subunit and change their apparent masses from 235 and 220 kDa to 159 kDa upon reduction of disulfide bonds. Nucleic acid hybridization with alpha 2 cDNA revealed an 8.0-kilobase transcript in carp skeletal muscle. Evidence for a copurification of subunits similar in size to mammalian beta or gamma subunits was not obtained.
Project description:Liver cytosol from mice fed on a normal diet contains Alpha-class glutathione S-transferase (GST) subunits of Mr 25,800, Mu-class GST subunits of Mr 26,400 and Pi-class GST subunits of Mr 24,800. Feeding female mice with a diet containing the anticarcinogenic antioxidant butylated hydroxyanisole (BHA) causes induction of the constitutively expressed Mu-class and Pi-class subunits. BHA also induces an Alpha-class GST comprising subunits of Mr 25,600, which is not expressed at detectable levels in normal mouse liver [McLellan & Hayes (1989) Biochem. J. 263, 393-402]. Data are now presented that show that administration of the anticarcinogen beta-naphthoflavone (BNF), like BHA, induces the Alpha-class 25,600-Mr subunits but not the constitutive Alpha-class GST with subunits of Mr 25,800. The effects of BNF on expression of hepatic GST were studied in both DBA/2 and C57BL/6 mice; these studies revealed a preferential induction of the Alpha-class 25,600-Mr subunits and of the Pi-class 24,800-Mr subunits in those mice in possession of a functional Ah receptor. The BHA/BNF-inducible Alpha-class GST can be resolved into two separate, non-interconvertible peaks by reverse-phase h.p.l.c. Automated amino acid sequence analysis of CNBr-derived peptides from each of these h.p.l.c.-purified peaks showed that the peaks contained at least two very similar subunits. These have been named Ya1 and Ya2. The amino acid sequence of the Ya1 subunit was compared with sequences deduced from a genomic clone, lambda mYa1 (Daniel, Sharon, Tichauer & Sarid (1987) DNA 6, 317-324], and a cDNA clone, pGT41 [Pearson, Reinhart, Sisk, Anderson & Adler (1988) J. Biol. Chem. 263, 13324-13332]. Our data suggest that the Ya1 subunit represents the subunit encoded by the genomic clone, lambda mYa1. Sequence analysis of the constitutive Alpha-class Ya3 subunit (Mr 25,800) shows that, although it is a member of the same gene family as the Ya1 and Ya2 subunits, it represents a distinct sub-family of Alpha-class GST, containing subunits that are more similar to rat Yc. Our data indicate that, of these Alpha-class GST subunits, the two with Mr 25,600 (Ya1 and Ya2) are selectively induced by BHA or BNF in mouse liver; neither BHA nor BNF induces significantly the GST subunit with Mr 25,800 (Ya3).