Abstract
We have cloned and sequenced a 9.4-kilobase cDNA specifying a new 280-kDa protein interacting with the cytoplasmic tail of glycoprotein (Gp) Ibalpha and showing considerable homology to actin-binding protein 280 (ABP-280) and chicken retinal filamin. We term this protein human beta-filamin. The gene for beta-filamin localizes to chromosome 3p14.3-p21.1. beta-Filamin mRNA expression was observed in many tissues and in cultured human umbilical vein endothelial cells (HUVECs); only minimal expression was detected in platelets and the megakaryocytic cell line CHRF-288. Like ABP-280, beta-filamin contains an NH2-terminal actin-binding domain, a backbone of 24 tandem repeats, and two "hinge" regions. A polyclonal antibody to the unique beta-filamin first hinge sequence identifies a strong 280-kDa band in HUVECs but only a weak band in platelets, and stains normal human endothelial cells in culture and in situ. We have confirmed the interaction of beta-filamin and GpIbalpha in platelet and HUVEC lysates. In addition, using two-hybrid analysis with deletion mutants, we have localized the binding domain for GpIbalpha in beta-filamin to residues 1862-2148, an area homologous to the GpIbalpha binding domain in ABP-280. beta-Filamin is a new member of the filamin family that may have significance for GpIbalpha function in endothelial cells and platelets.
Highlights
Glycoprotein (Gp)1 Ib␣, GpIb, GpIX, and GpV are all members of the leucine-rich glycoprotein superfamily and form a complex in the platelet membrane [1]
The GpIb complex is tightly bound to the actin cytoskeleton via an interaction of GpIb␣ with actin-binding protein 280 (ABP-280) [3,4,5]; it can be presumed that a similar interaction occurs in endothelial cells (ECs)
The term “filamin” was introduced by Singer’s laboratory [17] to describe a 250-kDa protein isolated from chicken gizzard, normally present as a dimer [18], that is capable of inducing actin polymerization [19]
Summary
Glycoprotein; ABP, actin-binding protein; ABPL, ABP-like protein; CRF, chicken retinal filamin; EC, endothelial cell; EST, expressed sequence tag; HUVEC, human umbilical vein endothelial cell; STS, sequence-tagged site; TABP, truncated ABP; vWF, von Willebrand factor; PCR, polymerase chain reaction; RT, reverse transcriptase; PBS, phosphate-buffered saline; kb, kilobase pair(s); bp, base pair(s); nt, nucleotide(s); BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody. Two partial cDNA fragments of one species, termed actin-binding protein-like protein (ABPL), were cloned by PCR and mapped to chromosome 7; full-length cDNA has not been reported. We report the molecular cloning and full sequence of a new human homolog of chicken filamin, which, like ABP-280, associates with the cytoplasmic tail of GpIb␣. It is present in only small amounts in platelets, but in substantial quantities in ECs. It is present in only small amounts in platelets, but in substantial quantities in ECs We term this new protein human -filamin, reserving the term ␣-filamin for ABP-280
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