Clarin-1, Encoded by the Usher Syndrome III Causative Gene, Forms a Membranous Microdomain

doi:10.1074/jbc.M109.003160

Clarin-1, Encoded by the Usher Syndrome III Causative Gene, Forms a Membranous Microdomain

POSSIBLE ROLE OF CLARIN-1 IN ORGANIZING THE ACTIN CYTOSKELETON*

From the ^‡Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106-4965,
the ^§Case Center for Proteomics, Case Western Reserve University, Cleveland, Ohio 44106-4965,
the ^¶Department of Ophthalmology, University of Florida, Gainesville, Florida 32610-0284,
the ^‖Department of Otolaryngology Head & Neck Surgery, University Hospitals Case Medical Center, Case Western Reserve University, Cleveland, Ohio 44106-4965,
the **Department of Molecular Genetics, Folkhälsan Institute of Genetics, FIN-00014 Helsinki, Finland,
the ^‡‡Department of Medical Genetics, University of Helsinki, FIN-00014 Helsinki, Finland,
the ^§§Department of Ophthalmology, University of Helsinki, FIN-00014 Helsinki, Finland, and
the ^¶¶Helen Wills Neuroscience Institute, University of California, Berkeley, California 94720

² To whom correspondence should be addressed:
Dept. of Pharmacology, School of Medicine, Case Western Reserve University, Wood Bldg., 10900 Euclid Ave, Cleveland, OH 44106-4965.
Tel.: 216-368-5226; Fax: 216-368-1300; E-mail: yxi19{at}case.edu.

↵¹ Both authors contributed equally to this work.

Abstract

Clarin-1 is the protein product encoded by the gene mutated in Usher syndrome III. Although the molecular function of clarin-1 is unknown, its primary structure predicts four transmembrane domains similar to a large family of membrane proteins that include tetraspanins. Here we investigated the role of clarin-1 by using heterologous expression and in vivo model systems. When expressed in HEK293 cells, clarin-1 localized to the plasma membrane and concentrated in low density compartments distinct from lipid rafts. Clarin-1 reorganized actin filament structures and induced lamellipodia. This actin-reorganizing function was absent in the modified protein encoded by the most prevalent North American Usher syndrome III mutation, the N48K form of clarin-1 deficient in N-linked glycosylation. Proteomics analyses revealed a number of clarin-1-interacting proteins involved in cell-cell adhesion, focal adhesions, cell migration, tight junctions, and regulation of the actin cytoskeleton. Consistent with the hypothesized role of clarin-1 in actin organization, F-actin-enriched stereocilia of auditory hair cells evidenced structural disorganization in Clrn1^−/− mice. These observations suggest a possible role for clarin-1 in the regulation and homeostasis of actin filaments, and link clarin-1 to the interactive network of Usher syndrome gene products.

Footnotes

↵* This work was supported by the Hope for Vision Foundation and the Elden Foundation.
↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3.
↵⁴ A detailed characterization of hearing phenotype in Clrn1^−/− mouse has been published recently (56).
↵3 The abbreviations used are:

CLRN1

clarin-1 protein

CLRN1

human clarin-1 gene, mRNA or cDNA

Clrn1

mouse clarin-1 gene, mRNA or cDNA

CLRN1^N48K

human clarin-1 protein with Asn at position 48 replaced by Lys

CLRN1^N48K

human clarin-1 gene, mRNA or cDNA, with Asn at position 48 replaced by Lys

HA

influenza hemagglutinin

HEK293

human embryonic kidney 293 cell

mAb

monoclonal antibody

MeβCD

methyl-β-cyclodextrin

RPE

retinal pigmented epithelial

RT

reverse transcription

tet

tetracycline

PBS

phosphate-buffered saline

MES

4-morpholineethanesulfonic acid

Bis-Tris

2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol

PNGase F

peptide N-glycosidase F

Endo H_f

endoglycosidase H fused to maltose binding protein

P

postnatal day.
- Received April 2, 2009.
- Revision received April 22, 2009.