Cloning, expression, and functional studies of a non-integrin platelet receptor for type I collagen

T. M. Chiang, A. Rinaldy, Andrew Kang

Research output: Contribution to journalArticle

Abstract

A cDNA encoding a platelet receptor protein hat binds type 1 collagen was cloned from a human bone marrow cDNA phage library. Cloning was achieved through the use of degenerate oligonucleotide probes derived from the amino acid sequence of a CNBr fragment of the purified receptor proetein. The size of the inserted cDNA was determined to be 1.6 kb by restriction enzyme analyses and comparison with standards. The inserted cDNA sequence was determined by dideoxy chain termination reactions. A computer search revealed this protein to be unique. A prokaryotic expression system (pKK 223-3-65 cDNA) yielded a 54 kDa recombinant protein which was isolated and purified to apparent homogeneity on 7.5% SDS-PAGE. A eukaryotic expression vector (pCDNA3-65 cDNA) yielded a 65 kDa protein, recognized by monoclonal antibody specific for the receptor protein isolated from platelet membranes. Like the receptor protein purified from platelet membranes, the recombinant protein inhibits type I collageninduced platelet aggregation, ATP release, and adhesion of [14C]-serotonin-labeled platelets to type I collagen in a dose dependent manner. The recombinant protein did not inhibit type III collagen- and ADP-induced platelet aggregation or ATP release, indicating the protein is specificic for type I collagen. Computer analysis of the deduced amino acid sequence from the sequenced nucleotides is compatible with three spanning transmembrane domains for this protein. These results suggest that the recombinant protein is a platelet binding protein (receptor) for type I collagen.

Original languageEnglish (US)
JournalFASEB Journal
Volume10
Issue number6
StatePublished - Dec 1 1996

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Cloning
Platelets
Organism Cloning
Blood Platelets
Complementary DNA
Recombinant Proteins
Collagen Type I
Proteins
Platelet Aggregation
Adenosine Triphosphate
Agglomeration
Restriction Mapping
Collagen Type III
Oligonucleotide Probes
Membranes
Amino Acids
Protein Sequence Analysis
Bacteriophages
Gene Library
Adenosine Diphosphate

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

Cite this

Cloning, expression, and functional studies of a non-integrin platelet receptor for type I collagen. / Chiang, T. M.; Rinaldy, A.; Kang, Andrew.

In: FASEB Journal, Vol. 10, No. 6, 01.12.1996.

Research output: Contribution to journalArticle

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