MPP5 recruits MPP4 to the CRB1 complex in photoreceptors.
OBJECTIVE
Mutations in the human Crumbs homologue 1 (CRB1) gene are a frequent cause of Leber congenital amaurosis (LCA) and various forms of retinitis pigmentosa. CRB1 is thought to organize an intracellular protein scaffold in the retina that is involved in photoreceptor polarity. This study was focused on the identification, subcellular localization, and binding characteristics of a novel member of the protein scaffold connected to CRB1.


METHODS
To dissect the protein scaffold connected to CRB1, the yeast two-hybrid approach was used to screen for interacting proteins. Glutathione S-transferase (GST) pull-down analysis and immunoprecipitation were used to verify protein-protein interactions. The subcellular localization of the proteins was visualized by immunohistochemistry and confocal microscopy on human retinas and immunoelectron microscopy on mouse retinas.


RESULTS
A novel member of the scaffold connected to CRB1, called membrane palmitoylated protein (MPP) subfamily member 4 (MPP4), a membrane-associated guanylate kinase (MAGUK) protein, was identified. MPP4 was found to exist in a complex with CRB1 through direct interaction with the MPP subfamily member MPP5 (PALS1). 3D homology modeling provided evidence for a mechanism that regulates the recruitment of both homo- and heterodimers of MPP4 and -5 proteins to the complex. Localization studies in the retina showed that CRB1, MPP5, and MPP4 colocalize at the outer limiting membrane (OLM).


CONCLUSIONS
These data imply that MPP4 and -5 have a role in photoreceptor polarity and, by association with CRB1, pinpoint the cognate genes as functional candidate genes for inherited retinopathies.
Kantardzhieva
Albena
A
Netherlands Ophthalmic Research Institute, Amsterdam.
Gosens
Ilse
I
Alexeeva
Svetlana
S
Punte
Ingrid M
IM
Versteeg
Inge
I
Krieger
Elmar
E
Neefjes-Mol
Carla A
CA
den Hollander
Anneke I
AI
Letteboer
Stef J F
SJ
Klooster
Jan
J
Cremers
Frans P M
FP
Roepman
Ronald
R
Wijnholds
Jan
J
D000818:Animals; D015153:Blotting, Western; D016764:Cell Polarity; D004285:Dogs; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D008958:Models, Molecular; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009703:Nucleoside-Phosphate Kinase / Q000235:genetics / Q000378:metabolism*; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020798:Two-Hybrid System Techniques
C448939:CRB1 protein, human; D005136:Eye Proteins; D008565:Membrane Proteins; C477071:Mpp4 protein, mouse; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D009703:Nucleoside-Phosphate Kinase; C470950:MPP4 protein, human; C501307:MPP5 protein, human; C499979:Mpp5 protein, mouse