Molecular cloning of a novel platelet protein showing homology to the angiotensin II receptor C-terminal domain

Abstract

Oligoscreening of a cDNA library obtained from 4β-phorbol 12-myristate 13-acetate-stimulated human erythroleukemia (HEL) cells resulted in the isolation of a novel clone coding for a protein with a calculated molecular mass of 8110 Da. This protein of 71 amino acids shows significant homology to the carboxyl-terminal regulatory domain of angiotensin II type 1 receptors. The homology encompasses four regions of amino acid residues thought to serve as consensus sequences for phosphorylation by serine/threonine kinases such as protein kinase C, which are key mediators of intracellular signaling. Reverse transcription-polymerase chain reaction identified the transcript in human platelets, human megakaryocytic DAMI cells, and HEL cells. High stringency Northern blotting revealed a tissue-specific distribution of three transcript species, with predominant expression in skeletal muscle and pancreas. Rabbit anti-peptide antiserum was used to immunoblot protein lysates from washed resting platelets and from 4β-phorbol 12-myristate 13- acetate-stimulated DAMI and HEL cells. These immunoblots revealed the presence of an intense ≃8-kDa protein band in platelets and HEL cells and a faint band of identical size in DAMI cells.