Molecular Cloning and Characterization of Human Trabeculin-α, a Giant Protein Defining a New Family of Actin-binding Proteins

doi:10.1074/jbc.274.47.33522

Molecular Cloning and Characterization of Human Trabeculin-α, a Giant Protein Defining a New Family of Actin-binding Proteins*

From the Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115

Abstract

We describe the molecular cloning and characterization of a novel giant human cytoplasmic protein, trabeculin-α (M _r = 614,000). Analysis of the deduced amino acid sequence reveals homologies with several putative functional domains, including a pair of α-actinin-like actin binding domains; regions of homology to plakins at either end of the giant polypeptide; 29 copies of a spectrin-like motif in the central region of the protein; two potential Ca²⁺-binding EF-hand motifs; and a Ser-rich region containing a repeated GSRX motif. With similarities to both plakins and spectrins, trabeculin-α appears to have evolved as a hybrid of these two families of proteins. The functionality of the actin binding domains located near the N terminus was confirmed with an F-actin binding assay using glutathioneS-transferase fusion proteins comprising amino acids 9–486 of the deduced peptide. Northern and Western blotting and immunofluorescence studies suggest that trabeculin is ubiquitously expressed and is distributed throughout the cytoplasm, though the protein was found to be greatly up-regulated upon differentiation of myoblasts into myotubes. Finally, the presence of cDNAs similar to, yet distinct from, trabeculin-α in both human and mouse suggests that trabeculins may form a new subfamily of giant actin-binding/cytoskeletal cross-linking proteins.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) .
↵‡ To whom correspondence should be addressed: Dana Farber Cancer Institute, Harvard Medical School, 44 Binney St., Boston, MA 02115 Tel.: 617-632-3386; Fax: 617-632-4470; E-mail: drchen@shore.net.
↵2 C. Leung, D. Sun, M. Zheng, D. Knowles, and R. K. H. Liem, EMBL accession number AF150755.
Abbreviations:

DMEM

Dulbecco's modified essential medium

bp

base pair(s)

PIPES

1,4-piperazinediethanesulfonic acid

GST

glutathioneS-transferase

PAGE

polyacrylamide gel electrophoresis

BSA

bovine serum albumin

PBS

phosphate-buffered saline

ABD

actin binding domain

EST

expressed sequence tag

BPAG1

bullous pemphigoid antigen 1

MTBD

microtubule binding domain
- Received August 20, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.