Shirin M. Marfatia

Plasma Membrane Ca2+ ATPase Isoform 4b Binds to Membrane-associated Guanylate Kinase (MAGUK) Proteins via Their PDZ (PSD-95/Dlg/ZO-1) Domains

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 andN-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 (K D = 1.6 nm), whereas binding to PDZ3 was in the micromolar range (K D = 1.2 μm). 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 OF HUMAN ERYTHROCYTE P55 MEDIATES ITS BINDING TO THE CYTOPLASMIC CARBOXYL TERMINUS OF GLYCOPHORIN C: ANALYSIS OF THE BINDING INTERFACE BY IN VITRO MUTAGENESIS

The PDZ domain, also known as the GLGF repeat/DHR domain, is an ∼90-amino acid motif discovered in a recently identified family of proteins termed MAGUKs (membrane-associated guanylatekinase 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 Drosophiladiscs 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.

VAM-1: a new member of the MAGUK family binds to human Veli-1 through a conserved domain.

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.

Localization of Dlg at the mammalian neuromuscular junction

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.