Interaction of two actin-binding proteins, synaptopodin and alpha-actinin-4, with the tight junction protein MAGI-1.
In an attempt to find podocyte-expressed proteins that may interact with the tight junction protein MAGI-1, we screened a glomerulus-enriched cDNA library with a probe consisting of both WW domains of MAGI-1. One of the isolated clones contained two WW domain-binding motifs and was identified as a portion of the actin-bundling protein synaptopodin. In vitro binding assays confirmed this interaction between MAGI-1 and synaptopodin and identified the second WW domain of MAGI-1 to be responsible for the interaction. MAGI-1 and synaptopodin can also interact in vivo, as they can be immunoprecipitated together from HEK293 cell lysates. Another actin-bundling protein that is found in glomerular podocytes and shown to be mutated in an inheritable form of glomerulosclerosis is alpha-actinin-4. We show that alpha-actinin-4 is also capable of binding to MAGI-1 in in vitro binding assays and that this interaction is mediated by the fifth PDZ domain of MAGI-1 binding to the C terminus of alpha-actinin-4. Exogenously expressed synaptopodin and alpha-actinin-4 were found to colocalize along with endogenous MAGI-1 at the tight junction of Madin-Darby canine kidney cells. The interaction and colocalization of MAGI-1 with two actin-bundling proteins suggest that MAGI-1 may play a role in actin cytoskeleton dynamics within polarized epithelial cells.
Patrie
Kevin M
KM
Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, 48109-0650, USA.
Drescher
Andrew J
AJ
Welihinda
Ajith
A
Mundel
Peter
P
Margolis
Ben
B
D000185:Actinin / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004285:Dogs; D005455:Fluorescent Antibody Technique; D051528:Guanylate Kinases; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism*; D009703:Nucleoside-Phosphate Kinase / Q000378:metabolism*; D011485:Protein Binding
D017931:DNA Primers; D008840:Microfilament Proteins; C503967:SYNPO protein, human; D000185:Actinin; D009703:Nucleoside-Phosphate Kinase; D051528:Guanylate Kinases