Search

The MAGUKs (membrane-associated guanylate kinase homologues) constitute a family of peripheral membrane proteins that function in tumor suppression and receptor clustering by forming multiprotein complexes containing distinct sets of transmembrane, cytoskeletal, and cytoplasmic signaling proteins. Here, we report the characterization of the human vam-1 gene that encodes a novel member of the p55 subfamily of MAGUKs. The complete cDNA sequence of VAM-1, tissue distribution of its mRNA, genomic structure, chromosomal localization, and Veli-1 binding properties are presented. The vam-1 gene is composed of 12 exons and spans approx. 115 kb. By fluorescence in situ hybridization the vam-1 gene was localized to 7p15-21, a chromosome region frequently disrupted in some human cancers. VAM-1 mRNA was abundant in human testis, brain, and kidney with lower levels detectable in other tissues. The primary structure of VAM-1, predicted from cDNA sequencing, consists of 540 amino acids including a single PDZ domain near the N-terminus, a central SH3 domain, and a C-terminal GUK (guanylate kinase-like) domain. Sequence alignment, heterologous transfection, GST pull-down experiments, and blot overlay assays revealed a conserved domain in VAM-1 that binds to Veli-1, the human homologue of the LIN-7 adaptor protein in Caenorhabditis. LIN-7 is known to play an essential role in the basolateral localization of the LET-23 tyrosine kinase receptor, by linking the receptor to LIN-2 and LIN-10 proteins. Our results therefore suggest that VAM-1 may function by promoting the assembly of a Veli-1 containing protein complex in neuronal as well as epithelial cells.

The human homologue of the Drosophila discs large tumor suppressor protein (hDlg), a member of the membrane-associated guanylate kinase (MAGUK) superfamily, interacts with K(+) channels, N-methyl-d-aspartate receptors, calcium ATPase, adenomatous polyposis coli, and PTEN tumor suppressor proteins, and several viral oncoproteins through its PDZ domains. MAGUKs play pivotal roles in the clustering and aggregation of receptors, ion channels, and cell adhesion molecules at the synapses. To investigate the physiological basis of hDlg interactions, we examined the self-association state of full-length hDlg as well as defined segments of hDlg expressed as recombinant proteins in bacteria and insect Sf9 cells. Gel permeation chromatography of full-length hDlg revealed that the purified protein migrates as a large particle of size >440 kDa. Similar measurements of defined domains of hDlg indicated that the anomalous mobility of hDlg originated from its amino-terminal domain. Ultrastructural analysis of hDlg by low angle rotary shadow electron microscopy revealed that the full-length hDlg protein as well as its amino-terminal domain exhibits a highly flexible irregular shape. Further evaluation of the self-association state of hDlg using sedimentation equilibrium centrifugation, matrix-assisted laser desorption/ionization mass spectrometry, and chemical cross-linking techniques confirmed that the oligomerization site of hDlg is contained within its amino-terminal domain. This unique amino-terminal domain mediates multimerization of hDlg into dimeric and tetrameric species in solution. Sedimentation velocity experiments demonstrated that the oligomerization domain exists as an elongated tetramer in solution. In vitro mutagenesis was used to demonstrate that a single cysteine residue present in the oligomerization domain of hDlg is not required for its self-association. Understanding the oligomerization status of hDlg may help to explicate the mechanism of hDlg association with multimeric K(+) channels and dimeric adenomatous polyposis coli tumor suppressor protein. Our findings, therefore, begin to rationalize the role of hDlg in the clustering of membrane channels and formation of multiprotein complexes necessary for signaling and cell proliferation pathways.

In Caenorhabditis elegans, mutations in the lin-2 gene inactivate the LET-23 receptor tyrosine kinase/Ras/MAP kinase pathway required for vulval cell differentiation. One function of LIN-2 is to localize LET-23 to the basal membrane domain of vulval precursor cells. LIN-2 belongs to the membrane-associated guanylate kinase family of proteins. We have cloned and characterized the human homolog of LIN-2, termed hCASK, and Northern and Western blot analyses reveal that it is ubiquitously expressed. Indirect immunofluorescence localizes CASK to distinct lateral and/or basal plasma membrane domains in different epithelial cell types. We detect in a yeast two-hybrid screen that the PDZ domain of hCASK binds to the heparan sulfate proteoglycan syndecan-2. This interaction is confirmed using in vitro binding assays and immunofluorescent colocalization. Furthermore, we demonstrate that hCASK binds the actin-binding protein 4.1. Syndecans are known to bind extracellular matrix, and to form coreceptor complexes with receptor tyrosine kinases. We speculate that CASK mediates a link between the extracellular matrix and the actin cytoskeleton via its interaction with syndecan and with protein 4.1. Like other membrane-associated guanylate kinases, its multidomain structure enables it to act as a scaffold at the membrane, potentially recruiting multiple proteins and coordinating signal transduction.

Recent studies have begun to elucidate the localization of ion channels and receptors in central nervous system synapses. A family of proteins containing PDZ domains has been suggested to play essential roles in these processes. PSD-95 and chapsyn-110 have been implicated in the clustering of Shaker K+ channels and NMDA receptors in the mammalian brain, and Dlg plays a role in the clustering of Shaker K+ channels at the Drosophila neuromuscular junction (NMJ). We have explored whether Dlg might participate in mammalian NMJ organization. We demonstrate that Dlg is expressed in muscle and co-localizes with utrophin at the post-synaptic face of the mammalian NMJ. Dlg may therefore be important for establishing or maintaining the organization of protein complexes at the mammalian NMJ.

Plasma membrane Ca2+ ATPases are P-type pumps important for intracellular Ca2+ homeostasis. The extreme C termini of alternatively spliced "b"-type Ca2+ pump isoforms resemble those of K+ channels and N-methyl-D-aspartate receptor subunits that interact with channel-clustering proteins of the membrane-associated guanylate kinase (MAGUK) family via PDZ domains. Yeast two-hybrid assays demonstrated strong interaction of Ca2+ pump 4b with the PDZ1 + 2 domains of several mammalian MAGUKs. Pump 4b and PSD-95 could be co-immunoprecipitated from COS-7 cells overexpressing these proteins. Surface plasmon resonance revealed that a C-terminal pump 4b peptide interacted with the PDZ1 + 2 domains of hDlg with nanomolar affinity (KD = 1.6 nM), whereas binding to PDZ3 was in the micromolar range (KD = 1.2 microM). In contrast, the corresponding C-terminal peptide of Ca2+ pump 2b interacted weakly with PDZ1 + 2 and not at all with PDZ3 of hDlg. Ca2+ pump 4b bound strongly to PDZ1 + 2 + 3 of hDlg on filter assays, whereas isoform 2b bound weakly, and the splice variants 2a and 4a failed to bind. Together, these data demonstrate a direct physical binding of Ca2+ pump isoform 4b to MAGUKs via their PDZ domains and reveal a novel role of alternative splicing within the family of plasma membrane Ca2+ pumps. Alternative splicing may dictate their specific interaction with PDZ domain-containing proteins, potentially influencing their localization and incorporation into functional multiprotein complexes at the plasma membrane.

The PDZ domain, also known as the GLGF repeat/DHR domain, is an approximately 90-amino acid motif discovered in a recently identified family of proteins termed MAGUKs (membrane-associated guanylate kinase homologues). Sequence comparison analysis has since identified PDZ domains in over 50 proteins. Like SH2 and SH3 domains, the PDZ domains mediate specific protein-protein interactions, whose specificities appear to be dictated by the primary structure of the PDZ domain as well as its binding target. Using recombinant fusion proteins and a blot overlay assay, we show that a single copy of the PDZ domain in human erythrocyte p55 binds to the carboxyl terminus of the cytoplasmic domain of human erythroid glycophorin C. Deletion mutagenesis of 21 amino acids at the amino terminus of the p55 PDZ domain completely abrogates its binding activity for glycophorin C. Using an alanine scan and surface plasmon resonance technique, we identify residues in the cytoplasmic domain of glycophorin C that are critical for its interaction with the PDZ domain. The recognition specificity of the p55 PDZ domain appears to be unique, since the three PDZ domains of hDlg (human lymphocyte homologue of the Drosophila discs large tumor suppressor) do not bind the cytoplasmic domain of glycophorin C. Taken together with our previous studies, these results complete the identification of interacting domains in the ternary complex between p55, glycophorin C, and protein 4.1. Implications of these findings are discussed in terms of binding specificity and the regulation of cytoskeleton-membrane interactions.

The oriented peptide library technique was used to investigate the peptide-binding specificities of nine PDZ domains. Each PDZ domain selected peptides with hydrophobic residues at the carboxyl terminus. Individual PDZ domains selected unique optimal motifs defined primarily by the carboxyl terminal three to seven residues of the peptides. One family of PDZ domains, including those of the Discs Large protein, selected peptides with the consensus motif Glu-(Ser/Thr)-Xxx-(Val/Ile) (where Xxx represents any amino acid) at the carboxyl terminus. In contrast, another family of PDZ domains, including those of LIN-2, p55, and Tiam-1, selected peptides with hydrophobic or aromatic side chains at the carboxyl terminal three residues. On the basis of crystal structures of the PSD-95-3 PDZ domain, the specificities observed with the peptide library can be rationalized.

The human homologue (hDIg) of the Drosophila discs-large tumor suppressor (DIg) is a multidomain protein consisting of a carboxyl-terminal guanylate kinase-like domain, an SH3 domain, and three slightly divergent copies of the PDZ (DHR/GLGF) domain. Here have examined the structural organization of the three PDZ domains of hDIg using a combination of protease digestion and in vitro binding measurements. Our results show that the PDZ domains are organized into two conformationally stable modules one (PDZ, consisting of PDZ domains 1 and 2, and the other (PDZ) corresponding to the third PDZ domain. Using amino acid sequencing and mass spectrometry, we determined the boundaries of the PDZ domains after digestion with endoproteinase Asp-N, trypsin, and alpha-chymotrypsin. The purified PDZ1+2, but not the PDZ3 domain, contains a high affinity binding site for the cytoplasmic domain of Shaker-type K+ channels. Similarly, we demonstrate that the PDZ1+2 domain can also specifically bind to ATP. Furthermore, we provide evidence for an in vivo interaction between hDIg and protein 4.1 and show that the hDIg protein contains a single high affinity protein 4.1-binding site that is not located within the PDZ domains. The results suggest a mechanism by which PDZ domain-binding proteins may be coupled to ATP and the membrane cytoskeleton via hDlg.

PDZ domains (also known as DHR domains or GLGF repeats) are approximately 90-residue repeats found in a number of proteins implicated in ion-channel and receptor clustering, and the linking of receptors to effector enzymes. PDZ domains are protein-recognition modules; some recognize proteins containing the consensus carboxy-terminal tripeptide motif S/TXV with high specificity. Other PDZ domains form homotypic dimers: the PDZ domain of the neuronal enzyme nitric oxide synthase binds to the PDZ domain of PSD-95, an interaction that has been implicated in its synaptic association. Here we report the crystal structure of the third PDZ domain of the human homologue of the Drosophila discs-large tumour-suppressor gene product, DlgA. It consists of a five-stranded antiparallel beta-barrel flanked by three alpha-helices. A groove runs over the surface of the domain, ending in a conserved hydrophobic pocket and a buried arginine; we suggest that this is the binding site for the C-terminal peptide.

Dematin and protein 4.2 are peripheral membrane proteins associated with the cytoplasmic surface of the human erythrocyte plasma membrane. Isoforms of dematin and protein 4.2 exist in many nonerythroid cells. In solution, dematin is a trimeric protein containing two subunits of 48 kDa and one subunit of 52 kDa. Recent determination of the primary structure of the 52 kDa subunit of dematin showed that it contains an additional 22-amino acid sequence in the headpiece domain. An alignment of the 22-amino acid insertion sequence revealed that the 52 kDa subunit of dematin shares a novel 11-amino acid motif with protein 4.2. In this communication, we report that the conserved 11-amino acid motif in dematin52 and protein 4.2 contains a nucleotide binding P-loop. Direct binding of ATP is demonstrated to the glutathione S-transferase fusion proteins containing corresponding segments of dematin52 and protein 4.2 as well as to purified protein 4.2. The binding of ATP to the recombinant domains of dematin52 and protein 4.2 is specific, saturable, and of high affinity. The nucleotide specificity of the P-loop is restricted to ATP since no detectable binding was observed with GTP. These results show that the 11-amino acid motif provides an ATP binding site in dematin52 and protein 4.2. Although the functional significance of ATP binding is not yet clear, our findings open new perspectives for the function of dematin and protein 4.2 in vivo.

Human p55 is an abundantly palmitoylated phosphoprotein of the erythroid membrane. It is the prototype of a newly discovered family of membrane-associated proteins termed MAGUKs (membrane-associated guanylate kinase homologues). The MAGUKs interact with the cytoskeleton and regulate cell proliferation, signaling pathways, and intercellular junctions. Here, we report the complete intron-exon map of the human erythroid p55 gene (HGMW-approved symbol MPP1). The structure of the p55 gene was determined from cosmid clones isolated from a cosmid library specific for the human X chromosome. There is a single copy of the p55 gene, composed of 12 exons and spanning approximately 28 kb in the q28 region of the human X chromosome. The exon sizes range from 69 (exon 5) to 203 (exon 10) bp, whereas the intron sizes vary from 280 bp (intron 2) to approximately 14 kb (intron 1). The intron-exon boundaries conform to the donor/acceptor consensus sequence, GT-AG, for splice junctions. Several of the exon boundaries correspond to the boundaries of functional domains in the p55 protein. These domains include a SH3 motif and a region that binds to cytoskeletal protein 4.1. In addition, a comparison of the genomic and the primary structures of p55 reveals a highly conserved phosphotyrosine domain located between the protein 4.1 binding domain and the guanylate kinase domain. Finally, promoter activity measurements of the region immediately upstream of the p55 gene, which contains several cis-elements commonly found in housekeeping genes, suggest that a CpG island may be associated with the p55 gene expression in vivo.

The Drosophila discs large tumor suppressor protein, Dlg, is the prototype of a newly discovered family of proteins termed MAGUKs (membrane-associated guanylate kinase homologues). MAGUKs are localized at the membrane-cytoskeleton interface, usually at cell-cell junctions, where they appear to have both structural and signaling roles. They contain several distinct domains, including a modified guanylate kinase domain, an SH3 motif, and one or three copies of the DHR (GLGF/PDZ) domain. Recessive lethal mutations in the discs large tumor suppressor gene interfere with the formation of septate junctions (thought to be the arthropod equivalent of tight junctions) between epithelial cells, and they cause neoplastic overgrowth of imaginal discs, suggesting a role for cell junctions in proliferation control. A homologue of the Dlg protein, named Hdlg, has been isolated from human B lymphocytes. It shows 65-79% identity to Dlg in the different domains, and it binds to the cytoskeletal protein 4.1. Here, we report that the gene for lymphocyte Hdlg, named DLG1, is located at chromosome band 3q29. This finding identifies a novel site for a candidate tumor suppressor on chromosome 3.

Protein 4.1 is the prototype of a family of proteins that include ezrin, talin, brain tumor suppressor merlin, and tyrosine phosphatases. All members of the protein 4.1 superfamily share a highly conserved N-terminal 30-kDa domain whose biological function is poorly understood. It is believed that the attachment of the cytoskeleton to the membrane may be mediated via this 30-kDa domain, a function that requires formation of multiprotein complexes at the plasma membrane. In this investigation, synthetically tagged peptides and bacterially expressed proteins were used to map the protein 4.1 binding site on human erythroid glycophorin C, a transmembrane glycoprotein, and on human erythroid p55, a palmitoylated peripheral membrane phosphoprotein. The results show that the 30-kDa domain of protein 4.1 binds to a 12-amino acid segment within the cytoplasmic domain of glycophorin C and to a positively charged, 39-amino acid motif in p55. Sequences similar to this charged motif are conserved in other members of the p55 superfamily, including the Drosophila discs-large tumor suppressor protein. Our data provide new insights into how protein 4.1, glycophorin C, p55, and their non-erythroid homologues, interact with the cytoskeleton to exert their physiological effects.

The Drosophila discs large tumor suppressor protein, dlg, has been shown to regulate the growth of imaginal discs during embryogenesis [Woods, D. F. & Bryant, P. J. (1991) Cell 66, 451-464]. We cloned and sequenced the complete cDNA for a human B-lymphocyte 100-kDa protein that shares 60% amino acid identity with dlg. This human homologue of Drosophila discs large (hdlg) contains a C-terminal domain homologous to the known guanylate kinases, a src homology 3 region motif, and three dlg homology repeats. Two nonhomologous domains that can contain in-frame insertions result in at least four alternatively spliced isoforms of hdlg. Several hdlg RNA transcripts are widely distributed in human and murine tissues, and the protein is localized to regions of cell-cell contact. Protein 4.1, the defining member of a family that includes talin and merlin/schwannomin, has the same cellular localization as hdlg, and two sites within hdlg associate in vitro with the 30-kDa N-terminal domain of protein 4.1.

p55 is a palmitoylated peripheral membrane phosphoprotein of human erythrocytes. Primary structure of p55 includes a single copy of the SH3 motif, a COOH-terminal guanylate kinase domain, and an NH2-terminal domain of unknown function. Although the function of p55 is not known, interest in this component has been heightened by its similarity to the Drosophila tumor suppressor discs-large (dlg). In this report we provide evidence for the direct association of p55 with the NH2-terminal 30-kDa domain of protein 4.1, a key component of the erythroid membrane skeleton. In addition, p55 also binds to the cytoplasmic domain of glycophorin C, a transmembrane protein of red blood cells. We also provide evidence demonstrating the direct association of the 30-kDa domain of protein 4.1 with the cytoplasmic domain of glycophorin C. Taken together, these results suggest the existence of a novel ternary complex at the erythroid plasma membrane involving protein 4.1, p55, and glycophorin C. Since isoforms of protein 4.1, p55, and glycophorin C are present in many non-erythroid cells, the binding interactions may be prototypical of similar associations that modulate cytoskeletal-membrane linkage of broad significance.