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 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.
Kim
E
E
Howard Hughes Medical Institute, Massachusetts General Hospital, Harvard Medical School, Boston 02114, USA.
DeMarco
S J
SJ
Marfatia
S M
SM
Chishti
A H
AH
Sheng
M
M
Strehler
E E
EE
D017398:Alternative Splicing; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D027682:Cation Transport Proteins; D029721:Drosophila Proteins; D051528:Guanylate Kinases; D019476:Insect Proteins / Q000737:chemistry; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry; D009419:Nerve Tissue Proteins / Q000737:chemistry; D009703:Nucleoside-Phosphate Kinase / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry; D053499:Plasma Membrane Calcium-Transporting ATPases; D025521:Tumor Suppressor Proteins; D062826:Zonula Occludens-1 Protein
D027682:Cation Transport Proteins; D029721:Drosophila Proteins; D019476:Insect Proteins; D007527:Isoenzymes; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D025521:Tumor Suppressor Proteins; D062826:Zonula Occludens-1 Protein; C033652:postsynaptic density proteins; C070350:dlg1 protein, Drosophila; D009703:Nucleoside-Phosphate Kinase; D051528:Guanylate Kinases; D053499:Plasma Membrane Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases