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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 96 No. 3 (August 1), 2000:
pp. 794-799
PLENARY PAPER
Molecular characterization of a granulocyte
macrophage-colony-stimulating factor receptor  subunit-associated
protein, GRAP
Jiangling Tu,
Nicos Karasavvas,
Mark L. Heaney,
Juan Carlos Vera, and
David W. Golde
From the Program in Molecular Pharmacology and Therapeutics
and the Department of Medicine, Memorial Sloane-Kettering Cancer
Center, New York, NY.
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Abstract |
The granulocyte macrophage-colony-stimulating factor
receptor (GM-CSF-R) is a heterodimer composed of 2 subunits,  and
 , and ligand binding to the high-affinity receptor leads to
signalling for the multiple actions of GM-CSF on target cells. In order
to explore the role of the subunit in signalling, we used a
yeast-2-hybrid system to identify proteins interacting with the
intracellular domain of the GMR-. A cDNA encoding a predicted
protein of 198 amino acids, designated GRAP (GM-CSF
receptor subunit-associated protein), was isolated in experiments using the
intracellular portion of GMR- as bait. The interaction between GRAP
and GMR- was confirmed by coimmunoprecipitation in mammalian cells.
GRAP mRNA is widely expressed in normal human and mouse tissues and in
neoplastic human cell lines, but it is not restricted to cells or
tissues that express GM-CSF receptors. Three discrete GRAP mRNA species
were detected in human tissues and cells, with estimated sizes of 3.3, 3.1, and 1.3 kb. GRAP is highly conserved throughout evolution, and
homologues are found in yeast. The GRAP locus in Saccharomyces
cerevisiae was disrupted, and mutant yeast cells showed an
inappropriate stress response under normal culture conditions, manifested by early accumulation of glycogen during the logarithmic growth phase. GRAP is, therefore, a highly conserved and widely expressed protein that binds to the intracellular domain of GMR-, and it appears to play an important role in cellular metabolism.
(Blood. 2000;96:794-799)
© 2000 by The American Society of Hematology.
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Introduction |
Granulocyte macrophage-colony-stimulating factor
(GM-CSF) is an important regulator of the growth, differentiation, and
maturation of myeloid precursor cells, and it enhances the function of
mature neutrophils and mononuclear phagocytes.1 GM-CSF
exerts its effect by interacting with its cognate receptor on the cell
surface. The GM-CSF receptor is a heterodimer composed of 2 subunits,
 and .1-4 The mature and subunits are
glycoproteins of 85 and 120 kd, respectively, that span the plasma
membrane once. The isolated subunit (GMR-) binds GM-CSF with low
affinity (Kd approximately 2-5 nmol/L), whereas the
isolated subunit (GMR-) does not bind GM-CSF but participates in
the formation of an / complex that binds GM-CSF with high
affinity (Kd approximately 30-100 pmol/L).
Responsive hematopoietic cells express high-affinity receptors that
transduce signals leading to intracellular protein phosphorylation.5-7 Because neither of the subunits of the
GM-CSF receptor has intrinsic kinase activity, GM-CSF signal
transduction appears to involve activation of cytosolic tyrosine
kinases such as Lyn, Fes, and Jak2. Jak2 has been shown to associate
with the subunit of the GM-CSF receptor on ligand
binding.8-10 The subunit is central to signal
transduction propagated by high-affinity GM-CSF receptors, and
mutagenesis experiments have identified distinct intracellular domains
of the subunit required to activate MAP kinase and
fos/jun pathways.11 Although the
cytoplasmic domain of the subunit is required for subunit-mediated signaling, the precise role of the subunit in
signal transduction in cells expressing the high-affinity receptor is
not well understood. We previously found that the isolated subunit
was able to signal for increased glucose uptake in different systems,
including human melanoma cells that naturally express only low-affinity
receptors and Xenopus laevis oocytes injected with GMR- mRNA
to express the low-affinity receptor.12,13 Signaling from
the subunit for substrate transport through the facilitative
glucose transporters does not appear to involve activation of the usual
kinase pathways.12 Experiments in c-null mice, however,
indicated that GM-CSF was not able to stimulate glucose uptake in bone
marrow cells, implying that under those circumstances the low-affinity
GMR was unable to signal for transport.14
The mechanism by which signals may be delivered through the isolated
subunit is unknown, and no proteins, other than GM-CSF and GMR-,
have been shown to associate with GMR-. We used a yeast 2-hybrid
system to identify proteins that interact with GMR-, and we isolated
a novel cDNA designated GRAP (GM-CSF receptor subunit-associated protein). We propose
that GRAP may play a role in GM-CSF receptor subunit-mediated
signal transduction and that it also has an important function in
cellular metabolism.
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Materials and methods |
Materials
The enhanced chemiluminescence Western blot detection system,
[-32P]dCTP and secondary antibodies (horseradish
peroxidase-linked antirabbit and antimouse) were from Amersham
(Arlington Heights, IL). The random primer labeling kit was from
Boehringer (Indianapolis, IN). Human tissue and cancer cell line
Northern blots, mouse total RNA, ExpressHyb hybridization solution,
human -actin cDNA probe, pEGFP-C vector, monoclonal antibodies to
green fluourescent protein (GFP), leukocyte GAD library
and the human muscle cDNA library were from Clontech (Palo Alto, CA).
The Dextran-sulfate transfection kit and TNT Quick coupled in vitro
transcription translation kit were from Promega (Madison, WI).
Antibodies to GMR- were from Santa Cruz Biotechnology (Santa Cruz,
CA). Protein G beads and the Coomassie plus protein assay reagent were
from Pierce (Rockford, IL). COS cells were purchased from ATCC
(Rockville, MD), and Nytran + membranes were from Schleicher & Schuell
(Keene, NH).
Strains and yeast genetic methods
The following Saccharomyces cerevisiae strains were used in
this study: R846 MATa his3- 200 trp1-901 leu2-3,112 ade2
LYS2::(lexAop)4.HIS3
URA3::(lexAop)8.lacZ gal4?
gal80?, obtained from R. Rothstein (Columbia University, New York,
NY) and AMP109 MATa/MATa leu2/leu2 trip1/trip1 ura3/ura3 lys2/lys2
ho:LYS2), obtained from A. Mitchell (Columbia University). Escherichia coli strains HB101 and XL1-Blue were used as
plasmid hosts. Standard genetic methods were followed.15
Yeast cells were grown in medium YEP (1% yeast extract, 2%
bacto-peptone) or synthetic complete medium (SC) lacking appropriate
amino acid supplements to maintain selection for plasmids. Growth on
different carbon sources was scored as described
previously.16 To assess sporulation proficiency, diploid
cells were grown on YEP-2% glucose plates overnight, patched on
sporulation medium15 incubated at room temperature for 1 to
6 days, and then examined microscopically for the presence of asci.
Plasmids
The construct pLexA-GMR- was made by inserting the
C-terminal (last 54 amino acids) GMR- SmaI-BamHI
fragment, obtained by PCR, into the multicloning sites of
pBTM116.17 The PCR primers were: 5' primer,
5'-CGCGGATCCCGGGCTTTAAAAGGTTCCTTAGGATACAG-3'; 3'
primer, 5'-CGCGGATCCGTCGACTACACCCTCTGGGTCTCAGG-3'. The
construct pLexA-IL3R- was made in a similar manner using
the following primers: 5' primer,
5'-CGCGGATCCCGGGCTGCAGAAGGTATCTGGTGATGCAG-3'; 3'
primer, 5'-CGCGGATCCGTCGACTTCCTGGCAGCTTCGGACGA-3'. The
construct pEGFP-GRAP was made by cloning the
HindIII-BamHI PCR fragment of GRAP C-terminal region
(the last 73 amino acids) into the multicloning sites of pEGFP-C
(Figure 1A). The PCR primers used for
amplification were: 5' primer,
5'-CCCCAAGCTTGCGGCCGCGTCGACGAAATATG-3'; 3' primer, 5'-CGGGATCCCCTTGGCCATATCAACAACTC-3'. Sequences of clones
recovered from the 2-hybrid screen were determined by primer extension
from the GAL4 region.18
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| Fig 1.
Restriction maps of the GRAP gene, the
GRH gene, and related constructs.
Genes and constructs are described in "Materials and methods,"
and the direction of transcription in all maps is from left to right.
(A) GRAP gene-coding region is shown in solid shading. GAD
extends from residues 768 to 881. Both pGAD-GRAP and pEGFP-GRAP are
fused to GRAP at codon 126. (B) GRH gene-coding region
is shown in the shaded striped bar. URA3 gene is shown in the
striped bar.
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2-hybrid screen for GMR--interacting proteins
The yeast LexAop-lacZ reporter strain R846 was transformed
with LexAp DNA-binding hybrid pLexA-GMR-, which expresses the C-terminal end of GMR- (last 54 amino acids) fused to
LexA. A single transformant was selected to grow on SC-Trp medium and then was transformed with a human leukocyte GAD library that expresses fusions between the Gal4 activation domain (GAD) and human leukocyte cDNA. The transformants containing the 2 hybrids were selected on
SC-Trp-Leu-His plates plus 25 mmol/L 3-AT. Positive clones were further
selected for -galactosidase activity.19 The plasmid DNA
from the positive clones was recovered from yeast19 and amplified in the E coli strain HB101. Each plasmid from the
positive clones was retested for interaction with LexA-GMR-, LexA,
and LexA-lamin.20 The plasmids that interacted with
LexA-GMR- and not with LexA or LexA-lamin were mapped and sequenced.
A 0.2-kb open reading frame DNA (BglII fragment) isolated from
pGAD-GRAP was used as a probe to screen a human muscle cDNA library by
hybridization. A 1.9-kb EcoRI fragment was recovered, subcloned
into the pUC19 vector, and sequenced. Both DNA strands were sequenced.
Transfections and immunoprecipitations
COS cells were co-transfected with vector containing the EGFP-GRAP
fusion protein or vector pEGFP alone, with the expression vector
pMX-GMR- encoding the full-length GMR-.12 Cells were transfected with a dextran-sulfate method according to the
manufacturer's instructions. Proteins were extracted as described
previously21 using a sodium dodecyl sulfate (SDS)-free RIPA
buffer (150 mmol/L NaCl, 1% deoxycholine, 1% Triton X-100 and 50 mmol/L Tris, and pH 7.2). Protein concentration was determined
spectrophotometrically, and approximately 100 µg of total protein
extract was used for each immunoprecipitation experiment.
Anti-GM-CSFR- antibodies were added (dilution 1:100) and incubated
at 4°C for 2 hours, and this was followed by a 1-hour incubation
with protein G beads. After washing with RIPA buffer, the
immunoprecipitate was resuspended in SDS-polyacrylamide gel
electrophoresis (PAGE) sample buffer, fractionated by 10% SDS-PAGE,
and immunoblotted. The primary antibodies were polyclonal anti-GMR-
C-18 (against the C-terminal 18 amino acids) and monoclonal anti-GFP
antibody. Secondary antibodies were horseradish peroxidase-linked
antirabbit and antimouse immunoglobulin. Immunoprecipitated proteins
were detected by enhanced chemiluminescence.
Northern and dot blot analyses of GRAP in human and mouse
tissues
Human Northern blots and RNA master dot blots contained poly
A+ RNA (Clontech, Palo Alto, CA). For the mouse Northern
blot, 5 µg total RNA was fractionated on a 1% formaldehyde-agarose
gel and transferred to Nytran + membrane. A BglII fragment from
pGAD-GRAP or a BglI-BglII fragment of GRAP cDNA clone
(Figure 1A) was radiolabeled using a random primer kit and
[-32P]dCTP. The Northern and the dot blot membranes
were hybridized with ExpressHyb hybridization solution
(Clontech, Palo Alto, CA) containing 2 × 106 cpm/mL
radiolabeled probe and washed according to the manufacturer's protocol. Human -actin (Northern blot) and ubiquitin (dot blot) probes were used as controls. The blots were visualized by exposure of
Kodak X-Omat film (Eastman Kodak, Rochester, NY).
Disruption of the GRAP-related homologue in yeast
A BLAST search revealed a yeast homologue of GRAP (GRAP-related
homologue; GRH) located in the SPC1-ILV3 intergenic region. The
hypothetical protein, YJRO14W, weighs 22.5 kd, and the function is
unknown. The GRH gene in S. cerevisiae was disrupted at the chromosomal locus. Primers containing 5' and 3'
untranslated sequences were fused with 5' and 3'
flanking sequences of the URA3 gene, respectively (Figure
1B): The 5' primer was
5'-CACCCCAAGGAAACAGTTCAAGAGCTAAACTA- AAGAAAAGCATATTGCATAAAATGTTAAGAGAATTCATCGATATC- TAGATCTCGAGC-3'. The 3' primer was
5'-CTCCCAGAACGGTGCTATTACATATTTATGGATTGCTTACTTGGCAGCTCCTTCTGCAGCAGG- CCAGTGAATTCCCGGGGATCCG-3'.
Using these 2 primers and pIC19R22 as a template, the PCR
product containing the URA3 gene and GRH1 flanking
sequences was used to disrupt the GRH locus of the wild-type diploid strain AMP109 selecting for uracil prototrophs.23
Tetrad analysis was performed by standard genetic
methods.15 The absence of the GRH gene in
haploid disrupters was confirmed by PCR analysis with primers
containing 5' and 3' flanking sequences: 5' primer, 5'-CGTGAACTTGCACCATGTACATC-3'; 3' primer,
5'-CTAACCTACACGCCTGGTTACCAAG-3'. The resultant allele was
designated grh1::URA3 (Figure 1B).
-galactosidase and glycogen assays
To test for -galactosidase expression, the transformants from the
positive clones were patched on selective SC-2% glucose plates, grown
for 1 day, replicated onto nitrocellulose filters, permeabilized
( 70°C for 10 minutes), and incubated with X-Gal overnight.24 To test for glycogen accumulation, yeast cells were plated onto YEP-2% glucose plates, incubated at 30°C
overnight, and stained with iodine vapor.25
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Results |
Identification of proteins that interact with GMR- using the
yeast 2-hybrid system
We used a yeast 2-hybrid system to isolate proteins that interact
with the cytoplasmic portion of GMR-. The construct LexA-GMR- was
used as "bait" to screen a human leukocyte cDNA library fused to
the GAD. From 2 million independent transformants, we isolated 107 positive clones that grew on SC-Trp-Leu-His plus 25 mmol/L 3-AT. Only 1 of the 107 clones activated the LexAop-lacZ reporter gene in
combination with the LexA-GMR- fusion protein. This positive clone
failed to activate the reporter lacZ gene in combination with
lexA or LexA-lamin (negative control; Table
1). Partial DNA sequencing from the GAD
junction revealed an in-frame fusion of a 0.2-kb open reading frame
fragment. We designated this new gene GM-CSF receptor -associated
protein (GRAP).
Because the GM-CSF and IL-3 receptors share the same subunit, we
tested whether GRAP associates with the subunit of the IL-3
receptor. We constructed a LexA-IL3R- fusion protein containing the
cytoplasmic portion of the IL3R- receptor (confirmed by Western blot
analysis; data not shown) and tested its association with the original
GAD-GRAP clone. We found that the IL3- receptor did not interact
with C-terminal 73aa of GRAP (Table 1), which indicates the selectivity
in the interaction of GRAP with the GMR-.
GRAP is a novel protein that interacts with GMR-
To clone the full-length cDNA of GRAP, we conducted a preliminary
Northern blot analysis that showed GRAP was highly expressed in
skeletal muscle. Therefore, we screened a human skeletal muscle cDNA
library using the original 0.2-kb GRAP DNA in the GAD clone as
a probe. A 1.9-kb cDNA clone was isolated and sequenced. An open
reading frame of 594 codons, encoding a putative 22.5-kd GRAP protein,
was identified. The original GAD clone corresponds to the C-terminal 73 amino acids of GRAP (Figure 1A). A GRAP cDNA clone was also isolated
from a cDNA library derived from the human myeloid leukemia cell line,
HL-60, and was identical to the GRAP open reading frame in the cDNA
clone of skeletal muscle library (data not shown).
BLAST sequence analysis revealed that GRAP is almost identical
to the human drp gene (Gene Bank accession number AF038554), which is described as a density-regulated protein.26 The
expression of drp was shown to increase in cultured cells at
high density. We identified 3 extra G residue positions at
50, 33, and 30 in the 5' region
upstream of GRAP compared with drp.
The cDNA sequence of GRAP contains 2 putative translational start sites
within the first 103 bp. The first ATG has a short open reading frame
of 22 amino acids. The second ATG is surrounded by nucleotide sequences
known to increase the translation initiation efficiency (Kozak
consensus sequences, 5'(A/G)NNATGG),27,28 and it is
followed by an open reading frame encoding a 198-amino acid protein
with an apparent molecular weight of 22.5 kd (Figure 2). In vitro translation of GRAP generated
a single band that migrated at approximately 30 kd on an SDS-PAGE gel
(data not shown). The disparity between the apparent and the expected
molecular weights of GRAP may be due to its amino acid composition.
GRAP is highly charged and such proteins, for example certain histones, can have atypical migration on SDS-PAGE gels.
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| Fig 2.
Sequence analysis of GRAP cDNA.
Nucleotide and amino acid numbers are indicated. The open reading frame
is shown in uppercase letters, and the 5'- and 3'-noncoding
regions are shown in lowercase letters. The atg and tag codons of the
small ORF are underlined. The Kozak consensus sequence is in bold.
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Evidence that GMR- and GRAP interact in vivo
To provide biochemical evidence of the interaction between GRAP and
GMR-, we tested whether GRAP and GMR- can be coimmunoprecipitated with antibodies that recognize the C-terminal portion of the GMR-. We first constructed a fusion protein (EGFP-GRAP) that contained the
last 73 amino acids of GRAP fused in-frame with the C terminal end of
enhanced green fluorescent protein (EGFP). COS cells cotransfected with
the plasmids pMX-GMR- and pEGFP-GRAP, or pMX-GMR- and pEGFP (control), were lysed under nondenaturing conditions. Cellular extracts
were collected and immunoprecipitated with anti-GMR- antisera. The
immunoprecipitated proteins were analyzed by Western blotting with
anti-GMR- and anti-GFP antibodies (Figure
3). An intense signal corresponding to
EGFP-GRAP fusion protein was detected in COS cells cotransfected with
pEGFP-GRAP and pMX-GMR- (Figure 3A). When the pEGFP-GRAP vector was
replaced by pEGFP, only a weak background signal corresponding to
EGFP was identified that likely represented nonspecific binding between
EGFP and GMR-. The coimmunoprecipitation of GMR- protein and the
fusion protein EGFP-GRAP supported the notion that GRAP and GMR-
associate in vivo. The immunoprecipitation data also confirmed the
interaction between GMR- and GRAP observed in the yeast 2-hybrid
system. In parallel experiments, anti-IL3R- antibodies failed to
coprecipitate EGFP-GRAP, suggesting that the interaction between GRAP
and GMR- is selective (data not shown).
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| Fig 3.
Interaction of GMR- with GRAP.
COS cells were cotransfected with plasmids encoding the wild type of
GMR- and the fusion protein EGFP-GRAP or EGFP as indicated. Cells
were lysed, and proteins were immunoprecipitated with anti-GMR-
sera. Immunoprecipitates were fractionated by 10% SDS-PAGE and
immunoblotted. Primary antibodies were polyclonal anti-GMR- and
monoclonal EGFP. The secondary antibody was horseradish
peroxidase-linked antirabbit or antimouse immunoglobulin. (A) Cell
lysates from cells transfected with GMR- and EGFP-GRAP or EGFP.
Immunoprecipitation with anti-GMR- and immunoblotting with anti
EGFP. (B) No immunoprecipitation. Immunoblotting was with anti-EGFP.
(C) Immunoprecipitation and immunoblotting with anti-GMR-.
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Expression of GRAP mRNA in human cells and tissues
Northern blots containing polyA+ RNA from a panel of
human cancer cell lines were hybridized with a 32P-labeled
GRAP probe (Figure 4). The analysis
revealed the presence of GRAP mRNA in myeloid leukemia cells (HL-60,
K-562, Molt4, and Raji) and also in tumor cell lines from ovary, colon,
lung, and melanoma (HeLa, SW480, A549, and G361) (Figure 4A). Three RNA species were detected in all cell lines a doublet at 3.1 and 3.3 kb
with intense hybridization signals and a fainter band at 1.3 kb. GRAP
mRNA was detected in cells known to express GM-CSF receptors, such as
HL-60, and was also found in cells lacking GM-CSF receptors, such as
Molt4 and K-562.29 Similarly, the expression of GRAP was
detected in normal human tissues, including spleen, thymus, prostate,
testis, ovary, small intestine, colon, and peripheral blood (Figure
4B). Because of the higher background on Northern blots from normal
human tissues, expression of the 1.3-kb mRNA species could not be
assessed. To investigate whether the 3 mRNA species detected on the
Northern blots encoded the same protein, we sequenced GRAP cDNA clones
isolated from HL-60 cells (3.0, 2.8, and 1.3 kb). All 3 clones had
identical ORF but variable 3' ends, suggesting that the GRAP mRNA
species on the Northern blots encoded the same protein but that
alternative splicing might have resulted in different mRNA species
(data not shown).
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| Fig 4.
Northern blot analysis and expression of GRAP mRNA in
tumor cell lines and normal human tissues.
Northern blots containing 2.5 µg human polyA+
RNA extracted from cancer cell lines or normal human tissues (panels A
and B, respectively). Blots were hybridized with GRAP sequences. RNA
size markers are shown on the left. Panels A and B correspond to blots.
Panels C and D correspond to -actin controls.
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To obtain a more complete picture of the expression pattern of GRAP in
human tissues, we used a human multi-tissue dot blot containing
polyA+ RNA from 50 different normal human tissues. We found
that GRAP is widely expressed in all human tissues, though the
intensity of the signal is variable (Figure
5). To confirm the wide distribution and
expression of GRAP in human tissues, we used the ORF of GRAP and searched the BCM search launcher web site (Human Genome Center, Baylor College of Medicine) for sequence similarities between GRAP and the database of Expressed Sequence Tags (dbEST). Human ESTs with high homology to GRAP (98%-100%) were expressed in
normal adult, embryonic, and neonatal tissues, and also in colon
carcinoma (Table 2). Based on the above
evidence, GRAP appears to be widely expressed in normal and neoplastic
tissues, and its expression is not restricted to cells and tissues
containing GM-CSF receptors.
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| Fig 5.
Human RNA master dot blot.
The human RNA dot blot contains polyA+ RNA extracted from
50 different human tissues. The dot blot was hybridized with GRAP
sequences. As a control, the same dot blot was hybridized with
ubiquitin cDNA sequences (middle panel). The dot blot diagram is shown
on the right panel.
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GRAP shares homology with proteins in mouse
Using the ORF of GRAP, we searched the BCM search launcher
web site for sequence similarities between GRAP and other ESTs. Several mouse EST sequences were homologous to GRAP (Table 2). The mouse ESTs were translated, and the consensus mouse GRAP
sequence was aligned with the human GRAP protein. As shown in Figure
6, the human and mouse GRAP proteins are
highly conserved with 96% identity. The sequence similarity between
GRAP and its mouse homologue was confirmed by Northern blot analysis.
Total RNA from several mouse tissues and HL-60 cells (positive control)
was transferred to a nylon membrane and hybridized with human GRAP
sequences (nucleotides 71-673). The human GRAP sequences hybridized
with a mouse homologue of GRAP message (mGRAP) (Figure 7). Mouse
tissues express a single mRNA band at approximately 1.3 kb. The 2 prominent bands of 3.3 and 3.1 kb, seen on the Northern blots
containing human RNA from HL-60 cells, are absent in the mouse,
suggesting that the shorter transcript of GRAP contains the entire ORF
(Figure 6).
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| Fig 6.
Amino acid alignment of GRAP in human, mouse, and yeast.
Shaded boxes indicate the amino acids that differ between the human and
mouse GRAP. The underlined sequence shows the part of GRAP that was
used to make the EGFP-GRAP fusion protein. Amino acid residues not
present in the GRAP sequence homologues are shown as dashes.
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Disruption of the GRAP homologue in S
cerevisiae causes early accumulation of glycogen
GRAP homologue genes are also found in S pombe and
S cerevisiae. At the protein level, the homology between GRAP,
S cerevisiae, and S pombe is 45% and 48%,
respectively. The predicted molecular weight of the S
cerevisiae GRAP homologue protein GRH is the same as the human,
22.5 kd, but its function in yeast is unknown. To determine the
phenotype of a GRH null mutation, we disrupted the GRAP
homologue locus in a wild-type diploid strain of SK-1 genetic background.30 Tetrad analysis of 2 disrupted diploids
heterozygous for the grh1::URA3 allele (Figure 1B),
showed 2:2 segregations for the URA3 marker. The grh1 mutant
showed no obvious defects in growth on glucose, galactose, or glycerol
at 30° or in growth on glucose at 37°. The mutants also showed
glucose-regulated invertase expression and sensitivity to the glucose
analogue 2-deoxyglucose (data not shown). To examine the effect on
sporulation, we crossed 2 grh1::URA3
mutant strains and found that the resultant mutant diploid strains
could sporulate. Iodine vapor stain for glycogen accumulation after
overnight growth showed 2:2 co-segregation with URA3 (Figure
8). The grh1 mutant
(URA3-positive) cells start to accumulate glycogen earlier. Overgrowing
cells (2-day incubation) showed little difference in glycogen
accumulation between wild-type and mutant cells (data not shown). Early
accumulation of glycogen in yeast lacking the GRAP homologue
represented an aberrant response to stress and suggested a role for
GRAP in glucose or glycogen metabolism.
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| Fig 7.
Northern blot analysis and expression of GRAP mRNA in
mouse tissues and HL-60 cells.
The upper panel shows the expression of GRAP in various mouse tissues.
Total RNA from human promyelocytic cells (HL-60) is used as a positive
control. RNA size markers are shown on the left. The lower panel shows
the same blot hybridized to actin sequences.
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| Fig 8.
Glycogen accumulation of wild-type and
mutant yeast cells with a disrupted GRAP homologue.
Five tetrads dissected from heterozygous diploids for the
grh1::URA3 allele were patched on the YED-2% glucose plate,
incubated at 30°C overnight, and stained with iodine vapor.
Dark-stained patches contain cells positive for glycogen accumulation
as well as the URA3 marker (data not shown).
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Discussion |
The cytoplasmic portion of the GMR- is important for
GM-CSF-mediated signaling through the high-affinity GM-CSF receptor and in certain systems the subunit appears capable of inducing increased uptake of glucose through the facilitative glucose
transporters. Proteins associating with GMR- that might mediate
these signals, however, have not been identified. We used a yeast
2-hybrid system to identify proteins interacting with GMR-. A cDNA
encoding a novel protein designated GRAP was isolated, and the protein
was shown to interact with the cytoplasmic segment of GMR-. This interaction was verified biochemically by coimmunoprecipitation experiments in which anti-GMR- antibody coprecipitated GMR- and
the EGFP-GRAP fusion protein. The isolation of GRAP, which directly
interacts with GMR-, offers an opportunity to explore signaling
mediated by GMR-.
A previously identified gene, drp, was isolated by differential
screening of a human teratocarcinoma cell line, PA-1; drp is a
partial clone thought to encode a 70-kd protein. Based on evidence
derived from our studies, which includes in vitro coupled transcription
translation, Kozak consensus sequence, and sequencing of cDNA clones
isolated from HL-60 cells representing mRNA species detected on
Northern blots, we believe that we have the entire ORF of GRAP. The
GRAP cDNA clone has an open reading frame of 198 amino acids and a
Kozak consensus sequence at position 3 to + 4 (gaaATGG). The
predicted molecular weight of GRAP is in agreement with the predicted
molecular weight of the S cerevisiae homologue. A GRAP
homologue identified in mouse tissues has 96% identity with the human
GRAP and lacks the 3.1- and 3.3-kb mRNA species. Therefore, the
smallest 1.3-kb mRNA detected in human and mouse Northern blots likely
contains the entire ORF. The significance of the 3.1- and 3.3-kb GRAP
messages in human cells is unknown. Analysis of the expression at the
RNA level revealed that GRAP is widely expressed and is present in all
tissues examined, though the degree of expression is variable. There is
a GRAP homologue in Saccharomyces, and GRAP mRNA is
expressed in cells that do not have GMR- (eg, K562 and IM-9 cells).
To elucidate the function of GRAP in vivo, we disrupted GRH in
S cerevisiae. The disruption did not interfere with normal yeast growth but did result in early accumulation of glycogen. Glycogen
accumulation in wild-type yeast cells starts when cells enter the
stationary phase, reflecting the stress response for yeast to nutrient
limitation. The early accumulation of glycogen in the grh1
mutant cells indicates an inappropriate stress response. Other known
mutant yeast cells with similar phenotype include mutation of genes
that cause defects in glucose signaling, such as reg1,
decreased cyclic adenosine monophosphate-dependent kinase activity,
such as ras2, cdc35/cyr1, and decreased glycogen
synthase activity, such as pho85. The role of GRH in the
regulation of glycogen accumulation requires further investigation,
however. Glycogen metabolism and signal transduction in mammals and
yeast share extensive similarities.31 We speculate that
GRAP may play a role in glucose or glycogen metabolism.
We have identified a novel protein that binds to the cytoplasmic
portion of GMR-. The GRAP protein is widely expressed in human
tissues and is highly conserved phylogenetically, indicating that its
function is not restricted to cells expressing GM-CSF receptors.
Phenotypic abnormalities characteristic of a stress response are
observed in yeast cells when the GRAP homologue is deleted. GRAP is the
first intracellular protein reported to interact directly with GMR,
though it is likely that the protein plays a wider role in cellular metabolism.
| |
Acknowledgments |
We thank Marian Carlson, Rodney Rothstein, and Aaron P. Mitchell for generously providing strains and plasmids, Maria Soushko for assisting with analysis of the GRH1 clones, and Nina
Chicharoen for technical assistance.
| |
Footnotes |
Submitted February 2, 2000; accepted March 20, 2000.
Supported by grants RO1 CA30388, RO1 HL42107, and P30 CA08748 from the
National Institutes of Health and by a research grant from the Leukemia
and Lymphoma Society.
J.T. and N.K. contributed equally to this work.
Reprints: David W. Golde, Memorial Sloan-Kettering Cancer
Center, 1275 York Avenue, New York, NY 10021; e-mail:
d-golde{at}ski.mskcc.org.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction