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

Oligoscreening of a cDNA library obtained from 4 beta-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 4 beta-phorbol 12-myristate 13-acetate-stimulated DAMI and HEL cells. These immunoblots revealed the presence of an intense approximately 8-kDa protein band in platelets and HEL cells and a faint band of identical size in DAMI cells.

Oligoscreening of a cDNA library obtained from 4␤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 4␤-phorbol 12-myristate 13-acetate-stimulated DAMI and HEL cells. These immunoblots revealed the presence of an intense Ϸ8-kDa protein band in platelets and HEL cells and a faint band of identical size in DAMI cells.
Cell growth and function are regulated through the action of a variety of extracellular ligands binding to specific receptors, initiating intracellular signaling mediated by sequentially activated protein kinases. Serine/threonine kinases such as protein kinase C are critically important enzymes modulating cellular function (1).
Platelets are the progeny of bone marrow megakaryocytes that circulate in blood to effect hemostasis and thrombosis. They are not only critically important for human physiology and pathology, but have also been used to study basic principles of cell signaling. Megakaryocytic cell lines such as HEL, 1 DAMI, and MEG-01 have been used to study megakaryocyte biology as well as to provide RNA from which cDNA libraries have been constructed and the cDNA of numerous platelet proteins cloned (2).
Angiotensin II (AII) is a potent effector octapeptide that mediates cardiovascular, neuronal, and renal function (3). Biological effects of AII are mediated by two distinct receptors, termed AT 1 and AT 2 . AT 1 receptors have been cloned from several mammalian sources (4 -9), and the AT 2 receptor has been cloned from rats (10,11). AII signaling in adult tissues is almost exclusively mediated by the AT 1 receptor (3). AT 1 and AT 2 receptors share an amino acid identity of 32% (10,11). The amphibian AII receptors that have been cloned are functionally similar to AT 1 receptors, but do not bind the non-peptide AIIbinding antagonist losartan (12,13). All AT 1 receptors and the amphibian AII receptors have several Ser/Thr residues in the carboxyl-terminal cytoplasmic tail that may serve as substrates for phosphorylation by protein kinase C (14). This phosphorylation may drive the agonist-induced internalization of AT 1 receptors (as well as other G protein-coupled receptors), resulting in cellular desensitization (15).
This report describes the isolation of a novel cDNA clone coding for a protein with a calculated molecular mass of 8110 Da. This protein shows significant homology to the regulatory C-terminal domains of the amphibian AII and mammalian AT 1 receptors (59 and 45%, respectively, over a 40-amino acid stretch). This homology includes four consensus protein kinase C phosphorylation sites and a receptor internalization signal (14,15). Immunoblotting with an anti-peptide antiserum demonstrates that the native protein is expressed in human platelets and cultured cells of human megakaryocytic lineage.

EXPERIMENTAL PROCEDURES
Isolation of the cDNA Clone-A 4␤-phorbol 12-myristate 13-acetatestimulated HEL cell line cDNA library in gt10 (CLONTECH) was oligoscreened simultaneously with two 30-mer oligoprobes (probe 1, 5Ј-GATGAACCICTICAGTCICCICCIGACCGI-3Ј; and probe 2, 5Ј-GAC-GAGCCICTICAGAGCCCICCIGACAGA-3Ј). The oligoprobes were based on the N-terminal sequence of a protein purified from shearactivated platelets (16). Phages (10 5 ) were screened, and one clone showed strong hybridization to the oligoprobes. Hybridization was done overnight at 55°C in 6 ϫ SSC, 0.5% SDS, 5 ϫ Denhardt's reagent, and 100 g/ml sonicated denatured salmon sperm DNA. The final wash was performed in 2 ϫ SSC, 0.5% SDS at 55°C for 30 min. Plaques were lifted on Colony/PlaqueScreen hybridization transfer membranes (Du-Pont NEN) and were treated as described by the manufacturer. Methods for bacterial and phage plating, labeling of oligoprobes, and purification were as described by Wallace and Miyada (17). Techniques for prehybridization and hybridization and other routine molecular biology methods were as described by Sambrook et al. (18).
Cloning and DNA Sequencing-One phage that showed the strongest hybridization to the oligoprobes was plaque-purified. Phage DNA was isolated by the method of Elliot and Green (19). This DNA was digested with EcoRI, and a 1.8-kilobase insert was cloned in pcDNAI (Invitrogen). DNA sequencing with purified recombinant plasmid (RP15a) was done with T7 and SP6 promoter primers and with internal sequence primers. A Sequenase kit (U. S. Biochemical Corp.) was used for plasmid sequencing.
Synthesis of all oligonucleotides was done by Operon Technologies, Inc. (Alameda, CA). Homology searches were done using EUGENE software at the Molecular Biology Computational Resource Center at * This work was supported by National Institutes of Health Grants HL02311 and HL02154 and by the Research Service of the Department of Veterans Affairs. The costs of publication of this article were defrayed in part by the payment of page charges. This 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 TM /EMBL Data Bank with accession number(s) L48211.
Baylor College of Medicine. The BLAST program of the National Center for Biotechnology Information was used for data base comparisons.
Northern Blotting-A human multiple tissue Northern blot panel was purchased from CLONTECH (Palo Alto, CA) and probed with labeled antisense RNA generated using SP6 RNA polymerase, [ 32 P]CTP, and the in vitro transcription kit. Prehybridization and hybridization were done in 6 ϫ SSC, 0.5% SDS, 5 ϫ Denhardt's reagent, and 100 g/ml sonicated denatured salmon sperm DNA at 65°C. Unincorporated label was removed using a Bio-Spin 30 column (Bio-Rad).
Immunoblotting-A polyclonal antiserum was prepared by immunizing rabbits with a synthetic peptide comprising putative amino acid residues 5-19 (FCSLGDRAACSVITA), a region without homology to any known proteins or cDNAs. The synthetic peptide and antiserum were prepared by Genosys Biotechnologies, Inc. (The Woodlands, TX).
Lysates from albumin gradient-washed platelets and from 100 ng/ml 4␤-phorbol 12-myristate 13-acetate-treated HEL and DAMI cells were prepared by adding to cells a solution containing 50% glycerol, 10% dithiothreitol, 8% SDS, and trace bromphenol blue, followed by immediate boiling for 10 min. SDS-polyacrylamide gel electrophoresis separations of 200 g of lysate protein were performed on 10 -20% gradient slab gels, and proteins were subsequently transferred to polyvinylidene difluoride membranes as described previously (20). Protein detection was carried out by established methods (18) using 3% skim milk in Tris-buffered saline as the blocking buffer. Goat anti-rabbit antibody conjugated to alkaline phosphatase (Life Technologies, Inc.) was used as the secondary antibody. In the competition experiments, 200 g/ml peptide was added to the primary antibody-containing solution.

RESULTS AND DISCUSSION
Analysis of the nucleotide sequence of the cloned HEL cell cDNA (Fig. 1) shows that the putative synthesis of a 71-amino acid protein (pRP15a) begins at an initiation codon following the Kozak rules for eukaryotic protein synthesis initiation (21). The 1815-base pair cDNA has the consensus polyadenylation signal sequence (AATAAA) 19 bases from the 3Ј-end and a selective mRNA destabilization signal (ATTTA) in the 3Ј-untranslated region (22). This signal is also present in the AII receptor 3Ј-untranslated sequences (see below).
Comparison of amino acid residues 21-60 of pRP15a with the C-terminal domain of angiotensin receptors reveals significant homology (Table I). pRP15a shares 34% identity and 59% homology (identical plus chemically similar amino acids) with the amphibian AII receptor. pRP15a shares an average identity of 26% and an average homology of 45% with members of the family of mammalian AT 1 receptors. The homology between pRP15a and AII receptors involves four potential consensus sequences for phosphorylation by Ser/Thr kinases such as protein kinase C (23). Hunyady et al. (15) have hypothesized that in the rat AT 1 receptor and other G protein-coupled receptors, phosphorylation at these Ser/Thr residues drives ligand-induced receptor internalization. More specifically, they have identified the sequence Ser-Thr-Leu in the C-terminal domain of the rat AT 1 receptor as crucial for receptor internalization. pRP15a has the sequence Ser-Phe-Leu (residues 32-34) (Table   FIG. 1. Nucleotide and deduced amino acid sequences of pRP15a. The consensus polyadenylation signal (AATAAA) is underlined. The ATTTA sequence is boxed. I) at these positions. The amphibian AII receptor also lacks the central Thr residue (Table I). Paxton et al. (24) have shown that the C-terminal 54 amino acids of the overexpressed rat AT 1 receptor serve as a substrate in vitro for the Src family of tyrosine kinases, and this tyrosine phosphorylation might regulate AT 1 receptor-induced activation of phospholipase C-␥ (25). pRP15a has tyrosine residues at positions 45 and 49, which are potential sites for phosphorylation by tyrosine kinases (Table I). The C-terminal 50 amino acids of the rat AT 1 receptor may also be required for receptor interactions with heterotrimeric GTP-binding proteins (26). The functional significance of the homology between pRP15a and the regulatory cytoplasmic C terminus of AII receptors remains to be established.
RT-PCR demonstrates that the transcript is present in platelets and in cultured platelet progenitor cell lines (Fig. 2). These results indicate that pRP15a gene expression occurs in cells of hematopoietic lineage (27).
High stringency Northern hybridization of a human multiple tissue blot (Fig. 3) shows that there is a tissue-specific distribution of transcripts of varying size. A transcript of Ϸ1.3 kilobases is predominant in skeletal muscle, and pancreas shows two transcript species of Ϸ1.65 and Ϸ1 kilobases. Brain and liver also have low level expression of these transcripts (data not shown). These three transcript sizes do not correspond to the length of the 1815-base pair cDNA clone and may therefore represent either alternative processing of a common RNA precursor or four different gene products with a high degree of homology.
In vitro translation of pRP15a using rabbit reticulocyte lysate (Life Technologies, Inc.) and [ 35 S]methionine produces a band with a molecular mass of Ϸ8 kDa as well as a series of smaller internal peptides that often mask the 8-kDa protein (data not shown). Immunoblotting of protein lysates shows an intense narrow band of Ϸ8 kDa in platelets and HEL cells and a faint band of identical size in DAMI cells. These bands are not seen on immunoblots performed in the presence of 200 g/ml immunizing peptide (Fig. 4).
pRP15a is likely to be a membrane-associated protein because this protein could not be expressed in bacteria using a vector under the control of a prokaryotic promoter (pBlue-Script). This necessitated the cloning of the 1.8-kilobase insert in a eukaryotic expression vector like pcDNAI. Hydropathy analysis revealed a single N-terminal hydrophobic domain, thereby providing additional evidence that pRP15a is membrane-associated (data not shown).
In summary, pRP15a appears to represent a novel protein that may serve as a substrate for phosphorylation by protein kinases. Its homology to the regulatory carboxyl-terminal regions of mammalian AT 1 and amphibian AII receptors supports the hypothesis that pRP15a may have some signaling function.
Further experiments are in progress to test this hypothesis by determining the location and phosphorylation state of the protein in resting and stimulated platelets, HEL cells, and DAMI cells and by examining the location and signaling function in heterologous cells overexpressing the recombinant protein.  2. Southern hybridization of RT-PCR products. RT-PCR was done with total RNA (ϳ2 g) isolated from platelets, DAMI cells, and HEL cells. The blot was hybridized with a labeled RNA probe prepared from RP15a as described under "Experimental Procedures." The final wash was in 0.1 ϫ SSC, 0.5% SDS at 65°C for 30 min. Autoradiography was for 24 h with a single intensifying screen. Molecular size markers in kilobases are shown to the left.  4. A, protein lysates from platelets and from 4␤-phorbol 12myristate 13-acetate-stimulated DAMI and HEL cells were subjected to SDS-polyacrylamide gel electrophoresis (10 -20% acrylamide), followed by transfer to polyvinylidene difluoride membranes. Membranes were blotted with rabbit antiserum to the synthetic peptide FCSLG-DRAACSVITA, corresponding to residues 5-19 of the putative translation product of RP15a. B, shown is immunoblotting in the presence of a 200 g/ml concentration of the synthetic peptide.