Interaction of the vascular cell adhesion molecule (VCAM-1) with its counter-receptor very late antigen-4 (VLA-4) (integrin ot4fl0 is important for a number of developmental pathways and inflammatory functions. We are investigating the molecular mechanism of this binding, in the interest of developing new anti-inflammatory drugs that block it. In a previous report, we showed that the predominant form of VCAM-1 on stimulated endothelial cells, seven-domain VCAM (VCAM-7D), is a functionally bivalent molecule. One binding site requires the first and the other requires the homologous fourth immunoglobulin-like domain. Rotary shadowing and electron microscopy of recombinant soluble VCAM-7D molecules suggests that the seven Ig-like domains are extended in a slightly bent linear array, rather than compactly folded together. We have systematically mutagenized the first domain of VCAM-6D (a monovalent, alternately spliced version missing domain 4) by replacing 3-4 amino acids of the VCAM sequence with corresponding portions of the related ICAM-1 molecule. Specific amino acids important for binding VLA-4 include aspartate 40 (D40), which corresponds to the acidic ICAM-1 residue glutamate 34 (E34) previously reported to be essential for binding of ICAM-1 to its integrin counter-receptor LFA-1. A small region of VCAM including D40, QIDS, can be replaced by the similar ICAM-1 sequence, GIET, without affecting function or epitopes, indicating that this region is part of a general integrin-binding structure rather than a determinant of binding specificity for a particular integrin. The VCAM-1 sequence G65NEH also appears to be involved in binding VLA-4. T ~E interaction of cells with one another via their membrane-bound adhesion molecules is a dynamic process with multiple physiologic and pathologic consequences. Molecular regulation of cellular adhesion and de-adhesion is responsible for modeling of the tissues and organs during development, and many of the same molecules continue to be important in both repair and normal function in the adult. Since its discovery a few years ago, the vascular cell adhesion molecule-1 (VCAM-1)l/very late antigen-4 (VLA-4) adhesion pathway has been shown to be involved in embryonic development of skeletal muscle (Rosen et al., 1992), and in hematopoiesis (Miyake et al., 1991), in addition to its originally described function as an inducible vascular adhesion pathway involved in migration of mononuAddress all correspondence to L. Osborn, Biogen, Inc., 14 Cambridge Center, Cambridge, MA 02142. C. Vassallo's current address is Amira Inc., Cambridge, MA. 1. Abbreviations used in this paper: VCAM-1, vascular cell adhesion molecule-l; VLA-4, very late antigen-4. clear leukocytes to sites of inflammation (Osborn et al., 1989; Elices et al., 1990). It has been implicated in the pathology of disorders affecting a wide range of processes, including metastasis of tumors (Rice and Bevilacqua, 1989), atherosclerosis (Cybulsky and Gimbrone, 1991), and autoimmune encephalitis (Yednock et al., 1992). VCAM-1 (also called INCAM-110) (Rice and Bevilacqua, 1989) is an Ig superfamily protein which is synthesized by endothelial cells in response to IL-1, TNF, LPS, or IL-4, and is found constitutively on a few other cell types, such as follicular dendritic cells in lymph nodes (Freedman et al., 1990) and bone marrow stromal cells (Miyake et al., 1991). The first counter-receptor identified for VCAM 1 was a member of the heterodimeric integrin family known as VLA-4 or a4~1, found on most mononuclear leukocytes, but not on neutrophils. VLA-4 can also bind to an alternately spliced site in fibronectin called CS-1 (Wayner et al., 1989; Guan and Hynes, 1990). The ct4 integrin chain in association with a different beta chain, a4f17 or ol4flp, appears to be capable of binding to both VCAM-1 and fibronectin (Ruegg et ai., © The Rockefeller University Press, 0021-95251941021601/8 $2.00 The Journal of Cell Biology, Volume 124, Number 4, February 1994 601-608 601 on A ril 3, 2017 D ow nladed fom Published February 15, 1994

We have determined the sequence and structure of the mouse Müllerian-inhibiting substance (MIS) type II receptor gene. Sequence comparisons demonstrate that the mouse, rat, rabbit, and human MIS type II receptors are highly conserved. The mouse MIS type II receptor gene is encoded by 11 exons and spans approximately 9-kb. Only half of the intron/exon boundaries of its kinase domain are conserved in comparison to the kinase domain of the related activin type II receptor. Whereas the activin type II receptor gene contains large introns (> 40-kb), the largest intron of the MIS type II receptor gene is only 4.3-kb. The MIS type II receptor gene (Amhr) is closely linked to Hoxc on mouse chromosome 15. Knowledge of the sequence and genomic structure of Amhr provides important information for the genetic manipulation of the Amhr locus.

Glial cell line-derived neurotrophic factor (GDNF)-dependent activation of the tyrosine kinase receptor RET is necessary for kidney and enteric neuron development, and mutations in RET are associated with human diseases, Activation of RET by GDNF has been shown to require an accessory component, GDNFR-alpha. (RETL1). We report the isolation and characterization of rat and human cDNAs for a novel cell-surface associated accessory protein, RETL2, that shares 49% identity with RETL1, Both RETL1 and RETL2 can mediate GDNF dependent phosphorylation of RET, but they exhibit different patterns of expression in fetal and adult tissues, The most striking differences in expression observed were in the adult central and peripheral nervous systems, In addition, the mechanisms by which the two accessory proteins facilitate the activation of RET by GDNF are quite distinct, In vitro binding experiments with soluble forms of RET, RETL1 and RETL2 demonstrate that while RETL1 binds GDNF tightly to form a membrane-associated complex which can then interact With RET, RETL2 only forms a high affinity complex with GDNF in the presence of RET. This strong RET dependence of the binding of RETL2 to GDNF was confirmed by FACS analysis on RETL1 and RETL2 expressing cells, Together with the recent discovery of a GDNF related protein, neurturin, these data raise the possibility that RETL1 and RETL2 have distinctive roles during development and in the nervous system of the adult, RETL1 and RETL2 represent new candidate susceptibility genes and/or modifier loci for RET-associated diseases.

Lymphotoxin-beta (LT-beta) is a member of the TNF family of ligands which when expressed with lymphotoxin-alpha (LT-alpha, i.e., the original LT or TNF-beta) forms a heteromeric complex with LT-alpha on the cell surface. The mouse gene structure was determined by both cDNA cloning and analysis of a genomic DNA fragment encompassing the TNF/LT locus in the H-2 region of chromosome 17. The mouse and human genomic structures were found to be similar in terms of location in the class III region of the MHC; however, the mouse gene lacks one intron found in most members of the family. Both the cDNA and the genomic sequences revealed an altered splice donor in the conventional intron 2 position, rendering it nonfunctional. The altered gene retains an open reading frame such that an additional 66 amino acids are inserted into the stalk region connecting the transmembrane domain with the receptor binding domain encoded by exon 4 in this type II membrane protein. Northern analysis showed that this gene is expressed predominantly in lymphoid organs. The outlining of the complete mouse TNF locus will further studies of the relationship between these genes and immune function.

Integrin alpha 4 beta 1 is a leukocyte receptor for fibronectin and vascular cell adhesion molecule-1 (VCAM-1). It is important in inflammatory recruitment of leukocytes, lymphopoiesis, and a number of development events. Here we have mapped a panel of functional monoclonal antibodies (mAbs) recognizing the integrin alpha 4 chain, using murine/human chimeric constructs expressed in COS7 cells. We find that: 1) mAbs that induce homotypic aggregation (epitope A mAbs) map to the most N-terminal 100 amino acids of the human alpha 4 chain; 2) mAbs that block adhesion of alpha 4 beta 1 to VCAM-1 and fibronectin (epitope B mAbs) map to a 52-amino-acid region between residues 152 and 203 of human alpha 4; 3) epitope B mAbs that do or do not induce aggregation (epitope B2 and B1 mAbs, respectively) map to the same regions and are therefore indistinguishable by this analysis; 4) mAbs that neither induce homotypic aggregation nor block adhesion (epitope C mAbs) map to a distinct region of the molecule comprising amino acids 422-606. The N-terminal region of the alpha 4 chain identified by functional A and B epitope mAbs does not correspond to ligand binding sites identified in other alpha subunits, such as cation binding sites or the "I-domain," which alpha 4 lacks, and thus represents a novel site for epitope functionality among the integrins.

DENDRITIC cells (DC) derived from bone marrow are critical in the function of the immune system, for they are the primary antigen-presenting cells in the activation of T-lymphocyte response. Their differentiation from precursor cells has not been defined at a molecular level, but recent studies have shown an association between expression of the relB subunit of the NF-kappa B complex(1-5) and the presence of DC in specific regions of normal unstimulated lymphoid tissues(4-6). Here we show that relB expression also correlates with differentiation of DC in autoimmune infiltrates in situ, and that a mutation disrupting the relB gene results in mice with impaired antigen-presenting cell function, and a syndrome of excess production of granulocytes and macrophages. Thymic UEA-1(+) medullary epithelial cells from normal mice show striking similarities to DC and, interestingly, these cells are also absent in relB mutant mice. Taken together, these results suggest that relB is critical in the coordinated activation of genes necessary for the differentiation of two unrelated but phenotypically similar cells (DC and thymic UEA-1(+) medullary epithelial cells) and is therefore a candidate for a gene determining lineage commitment in the immune system.

Efficient activation and regulation of the cellular immune response requires engagement of T cell accessory molecules as well as the antigen-specific T cell receptor. The lymphocyte function-associated antigen (LFA) 3 (CD58)/CD2 accessory pathway, one of the first discovered, has been extensively characterized in terms of structure and function of the CD2 molecule, which is present on all T lymphocytes and natural killer cells of the human immune system. The binding site of human CD2 for LFA-3 has been localized to two epitopes on one face of the first immunoglobulin (Ig)-like domain of this two-domain, Ig superfamily molecule. Human LFA-3 is genetically linked and is 21% identical in amino acid sequence to CD2, suggesting that this adhesive pair may have evolved from a single ancestral molecule. We have aligned the amino acid sequences of LFA-3 and CD2 and mutagenized selected amino acids in the first domain of LFA-3 that are analogous to those implicated in the binding site of CD2. The data show that K30 and K34, in the predicted C-C' loop, and D84, in the predicted F-G loop of LFA-3, are involved in binding to CD2, suggesting that two complementary sites on one face of the first domain of each molecule bind to each other.

Lymphotoxin (LT) -alpha beta heterotrimer is a membrane-anchored ligand expressed by activated T cells which binds specifically to the LT beta receptor (LT beta R), a member of the TNFR family. The LT beta R is implicated as a critical element in controlling lymph node development and cellular immune reactions. To address this hypothesis we have isolated a mouse cDNA encoding a single transmembrane protein of 415 amino acids with 76% identity to human LT beta R. The receptor function of this molecule was demonstrated by the ability of the extracellular domain, constructed as a chimera with the Fc region of IgG7, to bind to LT alpha beta complexes expressed on the surface of activated T cells or insect cells infected with baculoviruses containing LT alpha and LT beta cDNAs. The gene encoding mouse LT beta R, Ltbr, contains 10 exons spanning 6.9 kb and maps to mouse chromosome 6, which is closely linked to Tnfr1, consistent with the tight linkage of the human homologue of these genes on chromosome 12p13. Mouse LT beta R mRNA is expressed by cell lines of monocytic and epithelial origin but not by a CTL line, and in vivo it is constitutively expressed in visceral and lymphoid tissues. The delineation of the structure of the mouse LT beta R will aid investigations into the role of this cytokine-receptor system in immune function and development.

cDNA clones representing a small (0.8 kb) form of murine mucosal addressin cell adhesion molecule-1 (MAdCAM-1) mRNA were obtained and sequenced. The sequence was identical to the published 1.6 kb murine MAdCAM-1 cDNA sequence, except that 432 nucleotides encoding the mucin-like and IgA-homologous portions were deleted. This cDNA most likely represents an alternately spliced mRNA. Substantial amounts of both the short and long MAdCAM-1 mRNAs are present in murine mesenteric lymph node. Ig fusion proteins displaying either the short or long forms of MAdCAM-1 can bind Mn++-activated JY cells bearing human α4β7 integrin, indicating that the two N-terminal Ig-like domains of MAdCAM-1 are sufficient to bind its integrin counter-receptor.

Anti-Mullerian hormone (AMH) and its receptor are involved in the regression of Mullerian ducts in male fetuses. We have now cloned and mapped the human AMH receptor gene and provide genetic proof that it is required for AMH signalling, by identifying a mutation in the AMH receptor in a patient with persistent Mullerian duct syndrome. The mutation destroys the invariant dinucleotide at the 5' end of the second intron, generating two abnormal mRNAs, one missing the second exon, required for ligand binding, and the other incorporating the first 12 bases of the second intron. The similar phenotypes observed in AMH-deficient and AMH receptor-deficient individuals indicate that the AMH signalling machinery is remarkably simple, consisting of one ligand and one type II receptor.

We have isolated cDNA clones encoding the entire 170-kDa chain of epiligrin (alpha 3Ep) and a genomic clone encoding the alpha 3Ep gene (LamA3). Analysis of multiple cDNA clones revealed two distinct transcripts (alpha 3EpA and alpha 3EpB). Sequencing of the alpha 3EpA transcript indicated sequence and structural homology to laminin alpha 1 and alpha 2 chains that extend from domain IIIa through the carboxyl-terminal G domain. The alpha 3EpB transcript encodes a larger amino-terminal domain and contains additional epidermal growth factor repeats and sequences corresponding to domain IV of alpha 1 laminin. Fluorescence in situ hybridization indicated that the LamA3 gene is located on chromosome 18q11.2, a locus distinct from the LamA1 gene (18p11.3). The G domain of the epiligrin alpha 3 chain contains five subdomains that are individually related to the G subdomains reported for Drosophila and vertebrate laminin alpha chains. Sequence divergence within the G domain of alpha 3 epiligrin suggests that it is functionally distinct from laminin, consistent with our previous report showing that epiligrin interacts with different integrin adhesion receptors. Analysis of RNA from human foreskin keratinocytes (HFKs) identified multiple epiligrin transcripts that were down-regulated by viral transformation and differentiation. In contrast, epiligrin expression was up-regulated in wound sites of human skin.

Anti-Müllerian hormone, also called Müllerian-inhibiting substance or factor, is a glycoprotein dimer belonging to the transforming growth factor-beta superfamily and synthesized by immature Sertoli cells and postnatal granulosa cells. Anti-Müllerian hormone plays a key role in sex differentiation by inducing the regression of Müllerian ducts in the male fetus. It is also responsible for the stunting and masculinization of fetal ovaries in bovine freemartin fetuses and may be involved in the control of follicular maturation in the postnatal ovary. Using a degenerate probe for a consensus region of the transforming growth factor-beta receptor superfamily to screen a complementary DNA library from rabbit fetal ovaries, we cloned a complementary DNA coding for a transmembrane serine/threonine kinase, which is expressed around the fetal Müllerian duct, in fetal and adult granulosa cells, and in immature Sertoli cells. Two transcripts, generated by alternative splicing of an exon coding for an N-terminal 61-amino acid domain, are strongly expressed in anti-Müllerian hormone target organs and Sertoli cells. The longer, 569-amino acid, isoform binds anti-Müllerian hormone when transiently expressed in COS cells and is believed to encode its functional receptor.

Interaction of the vascular cell adhesion molecule (VCAM-1) with its counter-receptor very late antigen-4 (VLA-4) (integrin alpha 4 beta 1) is important for a number of developmental pathways and inflammatory functions. We are investigating the molecular mechanism of this binding, in the interest of developing new anti-inflammatory drugs that block it. In a previous report, we showed that the predominant form of VCAM-1 on stimulated endothelial cells, seven-domain VCAM (VCAM-7D), is a functionally bivalent molecule. One binding site requires the first and the other requires the homologous immunoglobulin-like domain. Rotary shadowing and electron microscopy of recombinant soluble VCAM-7D molecules suggests that the seven Ig-like domains are extended in a slightly bent linear array, rather than compactly folded together. We have systematically mutagenized the first domain of VCAM-6D (a monovalent, alternately spliced version mission domain 4) by replacing 3-4 amino acids of the VCAM sequence with corresponding portions of the related ICAM-1 molecule. Specific amino acids, important for binding VLA-4 include aspartate 40 (D40), which corresponds to the acidic ICAM-1 residue glutamate 34 (E34) previously reported to be essential for binding of ICAM-1 to its integrin counter-receptor LFA-1. A small region of VCAM including D40, QIDS, can be replaced by the similar ICAM-1 sequence, GIET, without affecting function or epitopes, indicating that this region is part of a general integrin-binding structure rather than a determinant of binding specificity for a particular integrin. The VCAM-1 sequence G65NEH also appears to be involved in binding VLA-4.

Two sCD4-resistant, primary viruses (P-08 and P-17) were compared with two sCD4-sensitive, T cell line-adapted variants (C-08 and C-17) for their biochemical responses to sCD4. At 37 degrees C, neither primary virus shed gp120 within 8 hr at sCD4 concentrations of up to 500 nM, whereas C-08 and C-17 lost gp120 within minutes of addition of 5-10 nM sCD4. At 4 degrees C, however, P-17 and C-17 shed gp120 at similar rates in response to the same sCD4 concentration. Irrespective of the temperature, gp120 dissociation from both P-17 and C-17 was inhibited by CD4 MAbs 6H10 and 5A8, the latter of which blocks events subsequent to sCD4 binding. Binding of sCD4 to P-17 was greater at 4 degrees C than at 37 degrees C, whereas the converse was found for C-17. Consistent with this, P-17 was neutralized much more potently by sCD4 at 4 degrees C than at 37 degrees C, whereas C-17 was slightly more sensitive to sCD4 at 37 degrees C than at 4 degrees C. Resistance to neutralization by sCD4 is probably determined by kinetic parameters. We suggest that the acquisition of sCD4 neutralization sensitivity and the above biochemical responses to sCD4 are coincidental to the process by which some primary viruses adapt to growth in transformed T cells. Sequence data indicate that there are a limited number of amino acid differences between the Env glycoproteins of the primary viruses and their T cell line-adapted counterparts; the significance of the individual changes is under investigation, but both pairs of viruses have amino acid substitutions in a region of gp41 thought to contact gp120.

The chemiluminescent detection methods described in this chapter have been successfully applied to the detection of plasmid DNA and genomic DNA in Southern and sequencing protocols. The high sensitivity and the simplicity of AMPPD are instrumental in making the chemiluminescent detection of DNA successful in hybridization assays. This detection technique has also been used to detect DNA in dot blots and in situ hybridization experiments as well as proteins in enzyme-linked immunosorbent assays (ELISAs) and Western blots.

Aminopeptidases catalyze the hydrolysis of amino acid residues from the amino terminus of peptide substrates. Leucine aminopeptidase (LAP) from bovine lens is the best characterized aminopeptidase and the only LAP for which the amino acid sequence was determined by protein sequencing. Using this sequence information, we isolated a bovine kidney LAP cDNA and compared its deduced amino acid sequence to the published amino acid sequence for bovine lens LAP. Overall, the sequences are highly conserved. However, several differences are observed. The kidney LAP cDNA indicates a 26 amino acid extension at the amino terminus which is not found in the mature purified lens LAP. The cDNA also indicates an additional octapeptide in the C-terminal region which was not indicated in the published lens LAP amino acid sequence but which was required for best fit of crystallographic data regarding bovine lens LAP. Several other single amino acid changes were also noted. Levels of LAP transcripts were examined in bovine lens and kidney tissue as well as in cultured lens cells. Lens epithelial tissue showed only one LAP transcript (2.4 kb) whereas two transcripts (2.0 and 2.4 kb) were observed in cultured lens cells derived from epithelial tissue and in kidney tissue. Using Northern blot analysis, we correlated LAP mRNA levels with previously determined changes of LAP activity in aging lens tissue and in progressively passaged lens epithelial cells which were used to simulate aging in vitro. No differences were found in LAP mRNA levels in epithelial tissue from old and young lenses.(ABSTRACT TRUNCATED AT 250 WORDS)