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Vol. 19, Issue 7, 2949-2961, July 2008

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The Endocytic Adaptor Protein ARH Associates with Motor and Centrosomal Proteins and Is Involved in Centrosome Assembly and Cytokinesis

Sanna Lehtonen^*,, Mehul Shah^*,, Rikke Nielsen^*, Noriaki Iino^*, Jennifer J. Ryan, Huilin Zhou^*,, and Marilyn G. Farquhar^*,

Departments of *Cellular and Molecular Medicine and Pathology, and Ludwig Institute for Cancer Research, University of California San Diego, La Jolla, CA 92093

Submitted June 4, 2007; Revised March 27, 2008; Accepted April 9, 2008
Monitoring Editor: Yixian Zheng

Numerous proteins involved in endocytosis at the plasma membrane have been shown to be present at novel intracellular locations and to have previously unrecognized functions. ARH (autosomal recessive hypercholesterolemia) is an endocytic clathrin-associated adaptor protein that sorts members of the LDL receptor superfamily (LDLR, megalin, LRP). We report here that ARH also associates with centrosomes in several cell types. ARH interacts with centrosomal (-tubulin and GPC2 and GPC3) and motor (dynein heavy and intermediate chains) proteins. ARH cofractionates with -tubulin on isolated centrosomes, and -tubulin and ARH interact on isolated membrane vesicles. During mitosis, ARH sequentially localizes to the nuclear membrane, kinetochores, spindle poles and the midbody. Arh^–/– embryonic fibroblasts (MEFs) show smaller or absent centrosomes suggesting ARH plays a role in centrosome assembly. Rat-1 fibroblasts depleted of ARH by siRNA and Arh^–/– MEFs exhibit a slower rate of growth and prolonged cytokinesis. Taken together the data suggest that the defects in centrosome assembly in ARH depleted cells may give rise to cell cycle and mitotic/cytokinesis defects. We propose that ARH participates in centrosomal and mitotic dynamics by interacting with centrosomal proteins. Whether the centrosomal and mitotic functions of ARH are related to its endocytic role remains to be established.

These authors contributed equally to this work.

Address correspondence to: Marilyn G. Farquhar (mfarquhar{at}ucsd.edu)

Abbreviations used: aa, amino acids; ARH, autosomal recessive hypercholesterolemia; dynein HC, dynein heavy chain; dynein IC, dynein intermediate chain; GCP2/GCP3, gamma-tubulin complex protein 2/3; GST, glutathione S-transferase; -TURC, -tubulin ring complex; IF, immunofluorescence; LDLR, low-density lipoprotein receptor; LRP, LDLR-related protein; MEF, mouse embryo fibroblast; PTB, phosphotyrosine binding; wt, wild type.