(Received for publication, April 4, 1995; and in revised form, October 31, 1995)
From the
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
8-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, ()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 and
AT
. AT
receptors have been cloned from several
mammalian
sources(4, 5, 6, 7, 8, 9) ,
and the AT
receptor has been cloned from
rats(10, 11) . AII signaling in adult tissues is
almost exclusively mediated by the AT
receptor(3) .
AT
and AT
receptors share an amino acid
identity of 32%(10, 11) . The amphibian AII receptors
that have been cloned are functionally similar to AT
receptors, but do not bind the non-peptide AII-binding antagonist
losartan(12, 13) . All AT
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
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 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.
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 Baylor College of Medicine. The BLAST program of the National Center for Biotechnology Information was used for data base comparisons.
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 µg 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 µg/ml
peptide was added to the primary antibody-containing solution.
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 3`-end and a selective mRNA destabilization signal (ATTTA) in the 3`-untranslated region(22) . This signal is also present in the AII receptor 3`-untranslated sequences (see below).
Figure 1: Nucleotide and deduced amino acid sequences of pRP15a. The consensus polyadenylation signal (AATAAA) is underlined. The ATTTA sequence is boxed.
Comparison of amino acid residues 21-60 of pRP15a with
the C-terminal domain of angiotensin receptors reveals significant
homology (Table 1). 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 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
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
receptor
as crucial for receptor internalization. pRP15a has the sequence
Ser-Phe-Leu (residues 32-34) (Table 1) at these positions.
The amphibian AII receptor also lacks the central Thr residue (Table 1). Paxton et al.(24) have shown that
the C-terminal 54 amino acids of the overexpressed rat AT
receptor serve as a substrate in vitro for the Src
family of tyrosine kinases, and this tyrosine phosphorylation might
regulate AT
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 1). The C-terminal 50 amino acids of the rat AT
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) .
Figure 2:
Southern hybridization of RT-PCR products.
RT-PCR was done with total RNA (2 µg) 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
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.
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 1.3 kilobases is predominant in skeletal
muscle, and pancreas shows two transcript species of
1.65 and
1
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.
Figure 3:
A,
a human multiple tissue Northern blot was probed with labeled antisense
RNA from RP15a as described under ``Experimental
Procedures.'' The blot was washed in 0.1 SSC, 0.5% SDS at
65 °C for 90 min. Autoradiography was done for 24 h with one
intensifying screen. Molecular size markers in kilobases are shown to
the left and right. B, the blot was stripped and reprobed with
human
-actin probe. The blot was washed in 0.1
SSC, 0.5%
SDS at 65 °C for 30 min. Autoradiography was done for 24 h with one
intensifying screen.
In vitro translation of pRP15a using rabbit
reticulocyte lysate (Life Technologies, Inc.) and
[S]methionine produces a band with a molecular
mass of
8 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
8 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
µg/ml immunizing peptide (Fig. 4).
Figure 4:
A, protein lysates from platelets and from
4-phorbol 12-myristate 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 FCSLGDRAACSVITA, corresponding to residues 5-19
of the putative translation product of RP15a. B, shown is
immunoblotting in the presence of a 200 µg/ml concentration of the
synthetic peptide.
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 (pBlueScript). 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 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L48211[GenBank].
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