Identification and Cloning of Centaurin-α

doi:10.1074/jbc.271.31.18859

Identification and Cloning of Centaurin-α

A NOVEL PHOSPHATIDYLINOSITOL 3,4,5-TRISPHOSPHATE-BINDING PROTEIN FROM RAT BRAIN*

From the ^‡ Neurobiology Research Center and the Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0021, the
^¶ Babraham Institute Laboratory of Molecular Signalling, Department of Zoology, Cambridge CB2 3ES, United Kingdom, the
^” Departments of Chemistry and Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, New York 11794-3400, and the
^# Howard Hughes Medical Institute, University of California at San Francisco, San Francisco, California 94143-0724

^∥
To whom correspondence should be addressed: Civitan International Research Center, Rm. 576A, 1719 Sixth Ave. South.
Tel.: 205-934-7278; Fax: 205-934-6571; E-mail: awtheibert{at}bmg.bhs.uab.edu

Abstract

Using an affinity resin and photoaffinity label based on phospholipid analogs of inositol 1,3,4,5-tetrakisphosphate (InsP₄), we have isolated, characterized, and cloned a 46-kDa protein from rat brain, which we have named centaurin-α. Binding specificity was determined using displacement of 1-O-[³H](3-[4-benzoyldihydrocinnamidyl]propyl)-InsP₄ photoaffinity labeling. Centaurin-α displayed highest affinity for phosphatidylinositol 3,4,5-trisphosphate (PtdInsP₃) (IC₅₀ = 120 nM), whereas InsP₄, PtdInsP₂, and InsP₃ bound with 5-, 12-, and >50-fold lower affinity, respectively. Screening a rat brain cDNA library with a polymerase chain reaction product, generated using partial amino acid sequence from tryptic peptides, yielded a full-length clone. The 2,450-base pair cDNA contained an open reading frame (ORF) encoding a novel protein of 419 amino acids. Northern analysis revealed a 2.5-kilobase transcript that is highly expressed in brain. The deduced sequence contains a novel putative zinc finger motif, 10 ankyrin-like repeats, and shows homology to recently identified yeast and mammalian Arf GTPase-activating proteins. Given the specificity of binding and enrichment in brain, centaurin-α is a candidate PtdInsP₃ receptor that may link the activation of phosphoinositide 3-kinase to downstream responses in the brain.

Footnotes

↵^§ Supported by a Comprehensive Minority Faculty Development program fellowship from the University of Alabama at Birmingham and a National Institutes of Mental Health research supplement for underrepresented minorities.
↵^∥ Supported by the Medical Research Council.
↵^‴ Supported by a National Science Foundation predoctoral fellowship and a National Institutes of Health research supplement for underrepresented minorities.
^∥ Supported by a National Science Foundation predoctoral training grant.
↵* This work was supported in part by National Institutes of Mental Health Grants R29MH50102 and DDRC P50HD32901 (to A. B. T.). Work at Stony Brook was supported by National Institutes of Health Grant NS29632 (to G. D. P.). The first two authors contributed equally to this study. 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) U51013[GenBank].
↵¹ The abbreviations used are:

PI

phosphoinositide

InsP_n

inositol polyphosphate

Ins(1,4,5)P₃

inositol 1,4,5-trisphosphate

DAG

diacylglycerol

InsP₄

inositol 1,3,4,5-tetrakisphosphate

InsP₅

inositol pentakisphosphate

InsP₆

inositol hexakisphosphate

PtdIns(3)P

phosphatidylinositol 3-phosphate

PtdIns(4)P

phosphatidylinositol 4-phosphate

PtdIns(4,5)P₂

phosphatidylinositol 4,5-bisphosphate

PtdInsP₃

phosphatidylinositol 3,4,5-trisphosphate

TEAB

triethylammonium bicarbonate

[¹²⁵I]ASA-InsP₄

1-O-[¹²⁵I](3-[2-iodo-4-azidosalicylamidyl]propyl)-inositol tetrakisphosphate

[³H]BZDC-InsP₄

1-O-[³H](3-[4-benzoyldihydrocinnamidyl]propyl)-inositol tetrakisphosphate

BZDC-NHS

4-benzoyldihydrocinnamoyl N-hydroxysuccinimide ester

HPLC

high performance liquid chromatography

gPInsP₃

glycerophosphoinositol 3,4,5-trisphosphate

daPtdInsP₃

diacetylphosphatidylinositol 3,4,5-trisphosphate

gPInsP₂

glycerophosphoinositol 4,5-bisphosphate

GAP

GTPase-activating protein

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid

PAGE

polyacrylamide gel electrophoresis

PCR

polymerase chain reaction

bp

base pair(s)

kb

kilobase(s)

ORF

open reading frame.
↵² The chimaeric nature of centaurin-α and its homology to a family of GTPase-activating proteins is reminiscent of another distinct family of molecules named the chimaerins (5), and we have therefore named our protein after another chimaera, the centaur (half-man, half-horse) of Greek mythology.
↵³ E. Tall, G. Dorman, P. Garcia, S. Shah, G. D. Prestwich, and M. J. Rebecchi, submitted for publication.
- Received March 27, 1996.
- Revision received May 13, 1996.