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Published on: December 7, 2021
MPP5 recruits MPP4 to the CRB1 complex in photoreceptors
Albena Kantardzhieva1, Ilse Gosens, Svetlana Alexeeva
1Netherlands Ophthalmic Research Institute, Amsterdam.
Purpose:
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.
Insights
Researchers identified membrane palmitoylated protein 4 (MPP4) as a novel component of the Crumbs homologue 1 (CRB1) protein scaffold. This discovery sheds light on photoreceptor polarity and inherited retinopathies.
Area of Science:
- Ophthalmology
- Molecular Biology
- Genetics
Background:
- Mutations in the Crumbs homologue 1 (CRB1) gene are a significant cause of Leber congenital amaurosis (LCA) and retinitis pigmentosa.
- CRB1 plays a crucial role in maintaining photoreceptor polarity by organizing an intracellular protein scaffold within the retina.
Purpose of the Study:
- To identify novel components of the CRB1-associated protein scaffold.
- To determine the subcellular localization and binding characteristics of these novel proteins.
- To investigate the role of these components in photoreceptor structure and function.
Main Methods:
- Yeast two-hybrid screening was employed to identify proteins interacting with CRB1.
- Glutathione S-transferase (GST) pull-down assays and immunoprecipitation were used to confirm protein-protein interactions.
- Immunohistochemistry, confocal microscopy (human retina), and immunoelectron microscopy (mouse retina) were utilized for subcellular localization studies.
Main Results:
- A novel scaffold protein, membrane palmitoylated protein 4 (MPP4), a member of the membrane-associated guanylate kinase (MAGUK) family, was identified.
- MPP4 directly interacts with MPP subfamily member MPP5 (PALS1), forming a complex with CRB1.
- CRB1, MPP5, and MPP4 were found to colocalize at the outer limiting membrane (OLM) of the retina.
Conclusions:
- MPP4 and MPP5 are integral to the CRB1 scaffold and play a role in photoreceptor polarity.
- The identified CRB1-MPP4-MPP5 complex implicates these genes as functional candidates for inherited retinopathies.
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