Twitchin from molluscan catch muscle: primary structure and relationship between site-specific phosphorylation and mechanical function

The phosphorylation state of the myosin thick filament-associated mini-titin, twitchin, regulates catch force maintenance in molluscan smooth muscle. The full-length cDNA for twitchin from the anterior byssus retractor muscle of the mussel Mytilus was obtained using PCR and 5'rapid amplification of cDNA ends, and its derived amino acid sequence showed a large molecule ( approximately 530 kDa) with a motif arrangement as follows: (Ig)11(IgFn2)2Ig(Fn)3Ig(Fn)2Ig(Fn)3(Ig)2(Fn)2(Ig)2 FnKinase(Ig)4. Other regions of note include a 79-residue sequence between Ig domains 6 and 7 (from the N terminus) in which more than 60% of the residues are Pro, Glu, Val, or Lys and between the 7th and 8th Ig domains, a DFRXXL motif similar to that thought to be necessary for high affinity binding of myosin light chain kinase to F-actin. Two major phosphorylation sites, i.e. D1 and D2, were located in linker regions between Ig domains 7 and 8 and Ig domains 21 and 22, respectively. Correlation of the phosphorylation state of twitchin, using antibodies specific to D1 and D2, with mechanical properties suggested that phosphorylation of both D1 and D2 is required for relaxation from the catch state.     

Novel immunoglobulin-like transcripts in teleost fish encode polymorphic receptors with cytoplasmic ITAM or ITIM and a new structural Ig domain similar to the natural cytotoxicity receptor NKp44

Members of the immunoglobulin superfamily (IgSF) include a group of innate immune receptors located in the leukocyte receptor complex (LRC) and other small clusters such as the TREM/NKp44 cluster. These receptors are characterised by the presence of immunoglobulin domains, a stalk, a transmembrane domain, and a cytoplasmic region containing either an immunoreceptor tyrosine-based inhibitory motif (ITIM) or are linked to an adapter molecule with an activation motif (ITAM) for downstream signalling. We have isolated two carp cDNA sequences encoding receptors in which the extracellular Ig domain structurally resembles the novel V-type Ig domain of NKp44. This is supported by a homology model. The cytoplasmic regions contain either an ITAM (Cyca-NILT1) or ITIMs (Cyca-NILT2). The tissue expression of these receptors is nearly identical, with the highest expression in the immunological organs. Peripheral blood leucocytes showed no detectable expression, but upon in vitro culture expressed NILT1, the activating receptor, and not the inhibitory NILT2 receptor. Southern blot analysis indicated that the NILT1 and NILT2 sequences belong to a multigene family. Analysis of the NILT Ig domain-encoding sequences amplified from both genomic DNA and cDNA revealed extensive haplotypic and allelic polymorphism. Database mining of the zebrafish genome identified several homologs on Chromosome 1, which also contains a cluster of class I major histocompatibility genes. This constellation is reminiscent of the TREM/NKp44 gene cluster and the HLA complex located on human Chromosome 6. The carp NILT genes form a unique cluster of innate immune receptors, which are highly polymorphic, and characterised by a new Ig structural subfamily and are distinct from the novel immune-type receptors (Nitrs) found in other fish species.     

Diversity of the killer cell Ig-like receptors of rhesus monkeys

Because the killer cell Ig-like receptors (KIRs) have only been characterized in humans and chimpanzees, we do not have a full understanding of their evolutionary history. Therefore, cDNAs encoding the KIR molecules of five rhesus monkeys were characterized, and were found to differ from the KIR molecules identified in humans and chimpanzees. Whereas only one KIR2DL4 molecule is detected in humans and chimpanzees, two distinct KIR2DL4 homologues were identified in the monkeys. Although the two human KIR3DL molecules are limited in their polymorphism, the KIR3DL homologues in the monkeys were highly polymorphic. Up to five KIR3DL homologues were identified in each monkey that was studied, and eleven distinct KIR3DL molecules were detected in the five rhesus monkeys. Two novel families of KIR molecules were identified in the rhesus monkeys, KIR3DH and KIR1D. The KIR3DH molecules have three Ig domains, transmembrane domains homologous to KIR2DL4 molecules that contain an arginine, and short cytoplasmic domains. With these features, the KIR3DH molecules resemble the activating forms of the human KIR molecules. The KIR1D molecule encodes only one complete Ig domain before a frame-shift in the second Ig domain occurs, leading to early termination of the molecule. Multiple splice variants of KIR1D exist that encode at least one Ig domain, as well as transmembrane and cytoplasmic domains. The extensive diversity of the rhesus monkey KIR3DL homologues and the novel KIR3DH and KIR1D molecules suggests that the KIR family of molecules has evolved rapidly during the evolution of primates.     

A multigene deletion in the human IGH constant region locus involves highly homologous hot spots of recombination

A simultaneous absence of the IgG1, IgG2, IgG4, and IgA1 immunoglobulins (Ig) was unambiguously demonstrated in six healthy individuals of two different families (family HASS and family TOU). These individuals were shown to be homozygous for a large deletion in the immunoglobulin heavy chain constant region locus. This deletion, which encompasses the G1-EP1-A1-GP-G2-G4 genes, allowed us to predict an order for the IgCH genes and to localize GP between A1 and G2. In this paper, we study the deletion-recombination point in the IGH locus of individual EZZ from the TOU family. We show that the distance between the G3 and the E genes on the EZZ recombinant chromosome is 24.7 kb and that the multigene deletion in the IgCH locus involves two highly homologous regions (hsg3 and hsg4) which are hot spots of recombination, outside of the switch sequences.     

Binding of leukemia inhibitory factor (LIF) to mutants of its low affinity receptor, gp190, reveals a LIF binding site outside and interactions between the two cytokine binding domains.

The gp190 transmembrane protein, the low affinity receptor for the leukemia inhibitory factor (LIF), belongs to the hematopoietin family of receptors characterized by the cytokine binding domain (CBD). gp190 is one of the very few members of this family to contain two such domains. The membrane-proximal CBD (herein called D2) is separated from the membrane-distal one (called D1) by an immunoglobulin-like (Ig) domain and is followed by three fibronectin type III repeats. We used truncated gp190 mutants and a blocking anti-gp190 monoclonal antibody to study the role of these repeats in low affinity receptor function. Our results showed that the D1Ig region was involved in LIF binding, while D2 appeared to be crucial for the proper folding of D1, suggesting functionally important interactions between the two CBDs in the wild-type protein. In addition, a point mutation in the carboxyl terminus of the Ig region strongly impaired ligand binding. These findings suggest that at least two distinct sites, both located within the D1Ig region, are involved in LIF binding to gp190, and more generally, that ligand binding sites on these receptors may well be located outside the canonical CBDs.     

The chicken leukocyte receptor complex: a highly diverse multigene family encoding at least six structurally distinct receptor types

The chicken Ig-like receptors (CHIR) have been described as two Ig domain molecules with long cytoplasmic tails containing inhibitory motifs. In this study, we demonstrate that CHIR form a large family, with multiple members showing great sequence variability among members as well as a great diversity in domain organization and properties of the transmembrane and cytoplasmic segments. We characterize various novel receptor types with motifs indicative of inhibitory, activating, or both functions. In addition to the inhibitory receptors with two ITIM, receptors with a single immunoreceptor tyrosine-based switch motif or receptors lacking a cytoplasmic domain were isolated. Activating receptors with a short cytoplasmic domain and a transmembrane arginine assembled with the newly identified chicken FcepsilonRIgamma chain. Three bifunctional receptor types were characterized composed of one or two C2-type Ig-like domains, a transmembrane region with a positively charged residue and combinations of cytoplasmic motifs such as ITIM, immunoreceptor tyrosine-based switch motif, and YXXM. RT-PCR revealed distinct expression patterns of individual CHIR. All receptor types shared a conserved genomic architecture, and in single Ig domain receptors a pseudoexon replaced the second Ig exon. Southern blot analyses with probes specific for the Ig1 domain were indicative of a large multigene family. Of 103 sequences from the Ig1 domain of a single animal, 41 unique sequences were obtained that displayed extensive variability within restricted Ig regions. Fluorescence in situ hybridization localized the CHIR gene cluster to microchromosome 31 and identified this region as orthologous to the human leukocyte receptor complex.     

Channel catfish leukocyte immune-type receptors contain a putative MHC class I binding site

The recent identification of a large and diverse family of leukocyte immune-type receptors (IpLITRs) in channel catfish (Ictalurus punctatus) indicates that immunoglobulin superfamily (IgSF) members related to both mammalian Fc receptors (FcRs) and leukocyte receptor complex (LRC)-encoded proteins exist in fish. In the present study, it was found that IpLITR messages were preferentially up regulated in catfish peripheral blood leukocytes (PBL) and clonal cytotoxic T cells (CTL) after alloantigen stimulation. Detailed sequence analyses of the expressed IpLITR cDNAs from two clonal CTL lines indicated an unexpectedly large array of putative activatory and inhibitory IpLITR-types containing variable numbers of extracellular immunoglobulin (Ig)-like domains. Importantly, all expressed IpLITRs shared similar membrane distal Ig domains (i.e., D1 and D2), suggesting that they may bind a common type of ligand. Sequence alignments and comparative homology modeling revealed that IpLITR domains, D1 and D2, have similar predicted 3-D structural properties with the corresponding domains of the human LRC-encoded leukocyte Ig-like receptor (LILR) family. Furthermore, conservation of key major histocompatibility class I (MHC I)-binding residues were located at similar positions within the membrane distal tip of D1 between representative IpLITRs and group 1 LILRs. Taken together, these results suggest that fish LITRs have an orthologous relationship to LRC-encoded receptors such as the human LILRs and could potentially function as a diverse family of MHC class I-binding receptors.     

Evolution of the human killer cell inhibitory receptor family

Phylogenetic analysis of different domains of human natural killer cell inhibitory receptors (KIR) implicated both intragenic duplication and deletion of exons and interlocus recombination in the evolution of these receptors. In phylogenies of the extracellular immunoglobulin (Ig) superfamily C2-set domains and of the pre-membrane (PM) domain, KIR receptors having two C2-set domains and those having three such domains tended to form separate clusters. However, the phylogenies of the transmembrane (TM) and cytoplasmic (CYT) domains showed quite different topologies, suggesting that major sites of interlocus recombination have been between exon 6 (encoding PM) and exon 7 (encoding TM) and between exon 7 and exons 8-9 (encoding CYT). Examination of the pattern of nucleotide substitution in the exons encoding Ig C2-set domains supported the hypothesis that positive Darwinian selection has acted to diversify the residues within these domains that are involved in contact with class I MHC molecules.     

Comparative analysis of the Kekkon molecules,related members of the LIG superfamily

Leucine-rich repeats (LRRs) and immunoglobulin (Ig) domains represent two of the most abundant sequence elements in metazoan proteomes. Despite this prevalence, comparatively few molecules containing both LRR and Ig (LIG) modules exist, and fewer still have been functionally defined. One LIG whose function has been investigated is the Drosophila protein Kekkon1 (Kek1). In vivo studies have demonstrated a role for Kek1 in Epidermal Growth Factor Receptor (EGFR) signaling and have suggested a role in neuronal pathfinding. Kek1 is the founding member of the Kek family, a group of six Drosophila transmembrane proteins that contain seven LRRs and a single Ig in their extracellular domains. While this arrangement of domains predicts a possible role as cell adhesion molecules (CAMs), to date little is known about the function or evolutionary relationship of these additional Kek molecules. Here we report that orthologs of Kek1, Kek2, Kek5, and Kek6 exist in the mosquito, Anopheles gambiae, and the honeybee, Apis mellifera, indicating that this family has been conserved for ~300 million years of evolutionary time. Comparative sequence analyses reveal remarkable identity among these orthologs, primarily in their extracellular regions. In contrast, the intracellular regions are more divergent, exhibiting only small pockets of conservation. In addition, we provide support for the general notion that these molecules may share common functions as CAMs, by demonstrating that Kek family members can form homotypic and heterotypic complexes.Edited by D. TautzChristina M. MacLaren, Timothy A. Evans and Diego Alvarado contributed equally to this work     

Structural insight into the specific interaction between murine SHPS-1/SIRP alpha and its ligand CD47

SRC homology 2 domain-containing protein tyrosine phosphatase substrate 1 (SHPS-1 or SIRPα/BIT) is an immunoglobulin (Ig) superfamily transmembrane receptor and a member of the signal regulatory protein (SIRP) family involved in cell-cell interaction. SHPS-1 binds to its ligand CD47 to relay an inhibitory signal for cellular responses, whereas SIRPβ, an activating member of the same family, does not bind to CD47 despite sharing a highly homologous ligand-binding domain with SHPS-1. To address the molecular basis for specific CD47 recognition by SHPS-1, we present the crystal structure of the ligand-binding domain of murine SHPS-1 (mSHPS-1). Folding topology revealed that mSHPS-1 adopts an I2-set Ig fold, but its overall structure resembles IgV domains of antigen receptors, although it has an extended loop structure (C′E loop), which forms a dimer interface in the crystal. Site-directed mutagenesis studies of mSHPS-1 identified critical residues for CD47 binding including sites in the C′E loop and regions corresponding to complementarity-determining regions of antigen receptors. The structural and functional features of mSHPS-1 are consistent with the human SHPS-1 structure except that human SHPS-1 has an additional β-strand D. These results suggest that the variable complementarity-determining region-like loop structures in the binding surface of SHPS-1 are generally required for ligand recognition in a manner similar to that of antigen receptors, which may explain the diverse ligand-binding specificities of SIRP family receptors.     

Identification of agonistic and antagonistic antibodies against gp190, the leukemia inhibitory factor receptor, reveals distinct roles for its two cytokine-binding domains

The receptor for the cytokine leukemia inhibitory factor (LIF) associates the low affinity binding component gp190 and the high affinity converter gp130, both of which are members of the family of hematopoietic receptors characterized by the cytokine receptor homology (CRH) domain. The gp190 is among the very few members of this large family to contain two CRH domains. The membrane-distal one (herein called D1) is followed by an Ig-like domain, a membrane-proximal CRH domain called D2, and three type III fibronectin repeats. We raised a series of monoclonal antibodies specific for the human gp190. Among them was the blocking antibody 1C7, which was directed against the D1Ig region and which impaired the binding of LIF to gp190. Another blocking antibody, called 12D3, was directed against domain D2 and interfered with the reconstitution of the high affinity receptor complex, independently of the interaction between LIF and gp190. The blocking effect of these two antibodies concerned four cytokines known to use gp190, i.e. LIF, oncostatin M, ciliary neurotrophic factor, and cardiotrophin-1. Among 23 antibodies tested alone or in combination (two anti-D2 and 21 anti-D1Ig), only the mixture of the two anti-D2 antibodies displayed agonistic activity in the absence of the cytokine. Taken together, these results demonstrate that the two CRH domains of gp190 play different functions in ligand binding and receptor activation.     

Molecular cloning and sequencing of cDNAs encoding the proteolipid subunit of the vacuolar H(+)-ATPase from a higher plant.

To understand the molecular structure of the vacuolar H(+)-translocating ATPase from plants, cDNAs encoding the N,N'-dicyclohexylcarbodiimide-binding 16-kDa proteolipid from oat (Avena sativa L. var. Lang) have been obtained. A synthetic oligonucleotide corresponding to a region of the bovine proteolipid cDNA (Mandel, M., Moriyama, Y., Hulmes, J.D., Pan, Y.-C.E., Nelson, H., and Nelson, N. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5521-5524) was used to screen an oat cDNA library constructed in lambda gt11. The nucleotide sequences of several positive clones (VATP-P1, clones 12, 54, 93) demonstrated the presence of a small multigene family. The four clones showed extensive divergence in their codon usage and their 3'-untranslated regions; however, the deduced amino acid sequences of the proteins were 97-99% identical. These clones encoded the proteolipid subunit as one of them (clone 12) expressed a fusion protein that reacted with an antibody to the 16-kDa proteolipid. The open reading frame of one cDNA clone (VATP-P1) predicted a polypeptide of 165 amino acids with a molecular mass of 16,641. Based on hydropathy plots, a molecule with four membrane-spanning domains was predicted, in which domain IV was especially conserved among different species. This domain showed 80% identity in nucleotide or amino acid sequences between the oat and the bovine proteolipids and contained a glutamate residue that is the putative N,N'-dicyclohexylcarbodiimide-binding residue. The presence of a small multigene family of the 16-kDa proteolipid was confirmed by Southern blot analysis showing that several distinct restriction fragments of oat nuclear DNA hybridized with the VATP-P1 cDNA.     

Sequence comparisons among dispersed members of the Brassica S multigene family in an S 9 genome

Self-incompatibility (SI) systems prevent self-pollination and promote outbreeding. In Brassica, the SI genes SLG (for S-locus glycoprotein) and SRK (for S-receptor kinase) are members of the S multigene family, which share the SLG-like domain (S domain), which encodes a putative receptor. We have cloned members of the S multigene family from the S9 haplotype of B. campestris (syn. rapa). In addition, eight distinct genomic regions harboring 10 SLG/SRK-like genes were characterized in the present study. Sequence analysis revealed two novel SRK-like genes, BcRK3 and BcRK6 (for B. campestris receptor kinases 3 and 6, respectively). Other genes that were characterized included SFR2 (for S gene family receptor 2), SLR2 (for S locus related gene 2), and a pseudogene. Based on phylogenetic analysis of the nucleotide sequences of the S domain regions, SLG and SRK appear to be distinct from other members of the S multigene family. Linkage analysis showed that most members of the S multigene family are dispersed in the Brassica genome, and that SLR1 (S locus related gene 1) is not linked to the SLR2 in B. campestris.     

Efficient neutralization and disruption of rhinovirus by chimeric ICAM-1/immunoglobulin molecules.

The intercellular adhesion molecule 1 (ICAM-1) is used as a cellular receptor by 90% of human rhinoviruses (HRVs). Chimeric immunoadhesin molecules containing extracellular domains of ICAM-1 and constant regions of immunoglobulins (Igs) were designed in order to determine the effect of increased valency, Ig isotype, and number of ICAM-1 domains on neutralization and disruption of rhinovirus structure. These immunoadhesins include ICAM-1 amino-terminal domains 1 and 2 fused to the hinge and constant domains of the heavy chains of IgA1, IgM, and IgG1 (IC1-2D/IgA, -/IgM, and -/IgG). In addition, all five extracellular domains were fused to IgA1 (IC1-5D/IgA). Immunoadhesins were compared with soluble forms of ICAM-1 containing five and two domains (sICAM-1 and ICI-2D, respectively) in assays of HRV binding, infectivity, and conformation. In prevention of HRV plaque formation, IC1-5D/IgA was 200 times and IC1-2D/IgM and IC1-2D/IgA were 25 and 10 times more effective, respectively, than ICAM-1. The same chimeras were highly effective in inhibiting binding of rhinovirus to cells and disrupting the conformation of the virus capsid, as demonstrated by generation of approximately 65S particles. The results show that the number of ICAM-1 domains and a flexible Ig hinge are important factors contributing to the efficacy of neutralization. The higher efficiency of chimeras that bound bivalently in disrupting HRV was attributed to higher binding avidity. The IC1-5D/IgA immunoadhesin was effective at nanomolar concentrations, making it feasible therapy for rhinovirus infection.     

The map1 gene of Cowdria ruminantium is a member of a multigene family containing both conserved and variable genes

Heartwater is an economically important disease of ruminants caused by the tick-transmitted rickettsia Cowdria ruminantium. The disease is present in Africa and the Caribbean and there is a risk of spread to the Americas, particularly because of a clinically asymptomatic carrier state in infected livestock and imported wild animals. The causative agent is closely related taxonomically to the human and animal pathogens Ehrlichia chaffeensis and Ehrlichia canis. A dominant immune response of infected animals or people is directed against variable outer membrane proteins of these agents known, in E. chaffeensis and E. canis, to be encoded by polymorphic multigene families. We demonstrate, by sequence analysis, that map1 encoding the major outer membrane protein of C. ruminantium is also encoded by a polymorphic multigene family. Two members of the gene family are located in tandem in the genome. The upstream member, orf2, is conserved, encoding only 2 amino acid substitutions among six different rickettsial strains from diverse locations in Africa and the Caribbean. In contrast, the downstream member, map1, contains variable and conserved regions between strains. Interestingly, orf2 is more closely related in sequence to omp1b of E. chaffeensis than to map1 of C. ruminantium. The regions that differ among orf2, map1, and omp1b correspond to previously identified variable sequences in outer membrane protein genes of E. chaffeensis and E. canis. These data suggest that diversity in these outer membrane proteins may arise by recombination among gene family members and offer a potential mechanism for persistence of infection in carrier animals.     

Mapping ligand binding domains in chimeric fibroblast growth factor receptor molecules. Multiple regions determine ligand binding specificity

Fibroblast growth factors (FGFs) mediate essential cellular functions by activating one of four alternatively spliced FGF receptors (FGFRs). To determine the mechanism regulating ligand binding affinity and specificity, soluble FGFR1 and FGFR3 binding domains were compared for activity. FGFR1 bound well to FGF2 but poorly to FGF8 and FGF9. In contrast, FGFR3 bound well to FGF8 and FGF9 but poorly to FGF2. The differential ligand binding specificity of these two receptors was exploited to map specific ligand binding regions in mutant and chimeric receptor molecules. Deletion of immunoglobulin-like (Ig) domain I did not effect ligand binding, thus localizing the binding region(s) to the distal two Ig domains. Mapping studies identified two regions that contribute to FGF binding. Additionally, FGF2 binding showed positive cooperativity, suggesting the presence of two binding sites on a single FGFR or two interacting sites on an FGFR dimer. Analysis of FGF8 and FGF9 binding to chimeric receptors showed that a broad region spanning Ig domain II and sequences further N-terminal determines binding specificity for these ligands. These data demonstrate that multiple regions of the FGFR regulate ligand binding specificity and that these regions are distinct with respect to different members of the FGF family.     

Evolution of plant Ash1 SET genes: structural divergence and functional differentiation

Plant Ash1 SET proteins are involved in H3K36 methylation, and play a key role in plant reproductive development. Genes encoding Ash1 SET proteins constitute a multigene family in which the copy number varies among plant species and functional divergence appears to have occurred repeatedly. To investigate the evolutionary history and functional differentiation of the Ash1 SET gene family, we made a comprehensive evolutionary analysis of this gene family from eleven major representatives of green plants. A novel deep sister relationship grouping previously resolved II-1 and II-2 orthologous groups was identified. The absence of AWS domain in the group II-2 suggests that the independent losses of AWS domain have occurred during evolution. A diversity of gene structures in plant Ash1 SET gene family have been presented since the divergence of Physcomitrella patens (moss) from the other land plants. A small proportion of codons in SET domain regions were detected to be under positive selection along the branches ancestral to land plant and angiosperms, which may have allowed changes of substrate specificity among different evolutionary groups while maintaining the primary function of SET domains. Our predictive subcellular localization and comparative anatomical meta-expression analyses can assort with the structural divergences of Ash1 SET proteins.     

The keratin BIIIB gene family: isolation of cDNA clones and structure of a gene and a related pseudogene

The nucleotide sequence of cDNA clones encoding the three major BIIIB high-sulfur wool keratin proteins (BIIIB2, 3, and 4) and the structure of a BIIIB4 gene and a BIIIB3 pseudogene are reported. Although Southern blot analysis indicates that the BIIIB genes comprise a multigene family in the sheep genome, they are poorly represented in genomic DNA libraries. The family sequence homology of the coding region extends into the 5' and 3' untranslated regions and the near 5' flanking region of the BIIIB3 and 4 genes. These homologies suggest that the BIIIB3 and 4 genes represent the latest gene duplication event in the evolution of the BIIIB multigene family. Like the genes coding for other wool keratin matrix protein components, the BIIIB genes have the conserved 18-bp sequence immediately 5' to the initiation codon and also appear to lack introns.     

Molecular analysis of receptor protein tyrosine phosphatase mu-mediated cell adhesion

Type IIB receptor protein tyrosine phosphatases (RPTPs) are bi-functional cell surface molecules. Their ectodomains mediate stable, homophilic, cell-adhesive interactions, whereas the intracellular catalytic regions can modulate the phosphorylation state of cadherin/catenin complexes. We describe a systematic investigation of the cell-adhesive properties of the extracellular region of RPTPmu, a prototypical type IIB RPTP. The crystal structure of a construct comprising its N-terminal MAM (meprin/A5/mu) and Ig domains was determined at 2.7 A resolution; this assigns the MAM fold to the jelly-roll family and reveals extensive interactions between the two domains, which form a rigid structural unit. Structure-based site-directed mutagenesis, serial domain deletions and cell-adhesion assays allowed us to identify the four N-terminal domains (MAM, Ig, fibronectin type III (FNIII)-1 and FNIII-2) as a minimal functional unit. Biophysical characterization revealed at least two independent types of homophilic interaction which, taken together, suggest that there is the potential for formation of a complex and possibly ordered array of receptor molecules at cell contact sites.