MGSA/GRO

26 Pages·0.646 MB·English

by  Wang D., Richmond A.

MGSA/GRO
Dingzhi Wang* and Ann Richmond
Department of Cell Biology, Vanderbilt University School of Medicine,
Department of Veterans Affairs, Nashville, TN 37232, USA
*corresponding author tel: [phone], fax: [phone],
e-mail: [email protected]
DOI: 10.1006/rwcy.2000.10001.
SUMMARY and nontumorigenic Chinese hamster embryonic
fibroblasts (CHEF/18), and the human GRO genes
MGSA/GRO is a CXC chemokine which shares were subsequently cloned (Anisowicz et al., 1987).
structuralfeaturesandmanybiologicalactivitieswith Since theoriginal isolationofhumanMGSA/GRO(cid:11),
IL-8. It is widely expressed in melanocytes, mela- two other isoforms have been identified, GRO(cid:12) and
noma, keratinocytes, monocytes and macrophages, GRO(cid:13) (Baker et al., 1990; Haskill et al., 1990; Iida
mammary epithelial cells, and endothelial cells. andGrotendorst,1990).Inaddition,afourthMGSA
Previously, MGSA/GRO was found to be important or GRO gene has recently been described, MGSA/
for recruitment and activation of neutrophils, lym- GRO(cid:14) (Shattuck-Brandt et al., 1997). However, we
phocytes,andmonocytesinhostdefense.Now,ithas havebeenunabletoverifytheexpressionofthisgene,
become apparent that MGSA/GRO plays an impor- which suggests that it may encode a pseudogene.
tant role in wound healing, growth regulation, angio-
genesis,tumorigenesis,andapoptosis.Theexpression Alternative names
of the MGSA/GRO ligands and receptors has been
detected in a number of inflammatory disorders and
The human MGSA/GRO(cid:11) gene has been described
in viral infections.
as MGSA(cid:11), GRO(cid:11), SCYB1, NAP-3, GRO1 onco-
gene and CXCL1; MGSA/GRO(cid:12) has been called
BACKGROUND MGSA(cid:12), GRO(cid:12), hMIP-2(cid:11), SCYB2, GRO2 onco-
gene, and CXCL2; and MGSO/GRO(cid:13) have been
referred to as MGSA(cid:13), GRO(cid:13), MIP-2(cid:12), SCYB3,
Discovery
GRO3oncogeneandCXCL3(Anisowiczetal.,1987;
Haskill et al., 1990; Tekamp-Olson et al., 1990;
Melanoma growth stimulatory activity (MGSA) was
Richmond and Shattuck, 1996). The murine MGSA/
first described as a secreted protein produced by
GRO homologs MIP-2 and KC have been cloned
malignant melanoma cells that functions as an auto-
(Oquendo et al., 1989; Cochran et al., 1983; Tekamp-
crine growth factor (Richmond et al., 1982, 1983,
Olson et al., 1990). MGSA/GRO(cid:14) is also referred to
1985).AfterpurifyingtheMGSAproteinfromserum-
as MGSA(cid:14). In rat, the MGSA or GRO mRNA are
free culture-conditioned medium produced by the
identified as CINC1, 2a, 2b, 3 (Huang et al., 1992a;
Hs294T human melanoma cell line (Richmond and
Konishi et al., 1993; Driscoll et al., 1993; Nakagawa
Thomas, 1986; Thomas and Richmond, 1988), a par-
et al., 1994).
tialaminoacidsequencewasdeterminedandthegene
wasclonedfromacDNA(Richmondetal.,1988).The
Structure
identified cDNA for MGSA(cid:11) exhibited 100% iden-
tity to the human homolog of the growth regulated
gene (GRO). GRO was first identified by subtractive The MGSA/GRO(cid:11) protein is translated as an
hybridization using mRNAs from tumorigenic 11,391Da precursor from which the signal peptide
Chinese hamster embryonic fibroblasts (CHEF/16) of 34 amino acids is cleaved to produce the mature
1024 Dingzhi Wang and Ann Richmond
protein of 7894Da (Balentien et al., 1990). Human acid level (Matsushima et al., 1988). MGSA/GRO
MGSA/GRO proteins are nonglycosylated proteins shares structural features and many biological acti-
which form dimers, tetramers, and larger aggregates vities with IL-8.
at high protein concentrations. At physiological con-
centrations (0.1–100nM), the monomer state pre- Main activities and
dominates (Fairbrother et al., 1993, 1994; Kim et al.,
pathophysiological roles
1994). The secondary structure of the monomer is
comprised of three (cid:12)-pleated sheet strands (residues
25–29, 39–44, and 49–52) and a C-terminal (cid:11) helix MGSA/GROplaysafundamentalroleinrecruitment
(residues 57–69). and activation of neutrophils, lymphocytes, and
All MGSA/GRO(cid:11), (cid:12), and (cid:13) proteins contain four monocytes in host defense. Numerous investigations
cysteines, an ELR motif at the N-terminus, and a have shown the importance of MGSA/GRO in acute
weakbindingdomainforheparinneartheC-terminus inflammation as chemotactic/activating factors for
(Clark-Lewis et al., 1993, 1994; Baggiolini et al., neutrophils, basophils, eosinophils, monocytes,
1994). These cysteines are involved in the formation smooth muscle cells, and lymphocytes (Balentien
of two intrachain disulfide bridges which are key for et al., 1990; Geiser et al., 1993; Loetscher et al., 1994;
maintenance of conformational integrity for receptor Schwartz et al., 1994; Yue et al., 1994; Erger and
binding(seeFigure2).Theglutamineresiduebetween Casale,1995;Jinquanetal.,1997;VanDammeetal.,
thefirsttwocysteinesisrequiredforCXCchemokine 1997). All three isoforms have the same pattern of
biological activities and receptor binding. This ELR activity, suggesting that the amino acid differences
motif is the critical structural/functional domain for among these isoforms result in only minor effects on
high-affinitybindingofIL-8,MGSA/GRO,ENA-78, biological activity. The order ofpotency for the three
GCP-2, KC, MIP-2, and other orthologs of this isoforms regarding neutrophil and basophil chemo-
familytotheCXCreceptors(Clark-Lewisetal.,1991, tactic activity, Ca2(cid:135) flux, respiratory burst, exocy-
1993, 1994; He´bert et al., 1991; Clubb et al., 1994; tosis, shape change, and receptor binding is MGSA/
Rajarathnam et al., 1994). CXC chemokines without GRO(cid:11)>(cid:13)>(cid:12) (Geiser et al., 1993). It has become
this motif do not bind to CXCR1 or CXCR2 (Clark- apparent that MGSA/GRO plays an important role
Lewis et al., 1993, 1994). When the ELR residues of in wound healing, growth regulation, angiogenesis,
MGSA/GRO or IL-8 are substituted with alanine, tumorigenesis, and apoptosis. The expression of the
biologicalactivityislostandthemutatedchemokines MGSA/GRO ligands and receptors has been impli-
act as antagonists in the presence of physiologic con- cated in a number of inflammatory disorders and in
centrations of ELR motif-containing chemokines viral infections (See Table 1).
(Arenbergetal.,1997;ZagorskiandWahl,1997).The
C-terminal portion of the human MGSA/GRO is
GENE AND GENE REGULATION
sufficient for its biological activity (Roby and Page,
1995). Mutation of the histidine residue (H19A) in
Accession numbers
MGSA/GROisassociatedwitha>100-folddecrease
in neutrophil chemotaxis, and pretreatment of cells
withtheH19AmutantinhibitedtheabilityofMGSA/ See Table 2.
GRO to induce elastase release and chemotaxis and
to increase intracellular calcium (Baly et al., 1998). Chromosome location
The chemokine MGSA/GRO is highly conserved
among multiple isoforms (see Relevant linkages).
MGSA/GRO(cid:11), (cid:12), and (cid:13) genes are located on human
MGSA/GRO(cid:12) and MGSA/GRO(cid:13) exhibit 93% and
chromosome 4q13-21, while the mouse orthologs are
82% nucleotide sequence identity to MGSA/GRO(cid:11).
located on chromosome 5 (Anisowicz et al., 1988;
In protein sequence, there are 11 amino acid differ-
Richmondetal.,1988;Seldinetal.,1990).Thebovine
ences between (cid:12) and (cid:11) and 15 amino acid differences
orthologs are located on chromosome 6, which is
between(cid:13) and(cid:11).Fourofthedifferencesareinthe34
consistent with the positions in the human (Modi
amino acid signal peptide, making only 7 and 11
et al., 1998).
amino acid differences in the mature 73 amino acid
secreted protein between the (cid:11) and (cid:12) or (cid:13) forms
Relevant linkages
(Haskill et al., 1990; Iida and Grotendorst, 1990;
Tekamp-Olson et al., 1990) (see Figure 2). The
identity between MGSA/GRO(cid:11) and IL-8 is about A subfamily of polypeptide chemoattractants known
42% at the nucleotide level and 56% at the amino as CXC chemokines is characterized by the ability to
MGSA/GRO 1025
Table 1 Human diseases associated with increased MGSA/GRO production
Inflammatory Psoriasis Schroder et al., 1992
Ulcerative colitis Isaacs et al., 1992
Rheumatoid arthritis Hosaka et al., 1994; Koch et al., 1995
Bacterial pneumonia Villard et al., 1995
Adult respiratory distress syndrome Villard et al., 1995
Helicobacter pylori infection Shimoyama and Crabtree, 1997;
Bodger and Crabtree, 1998
Intra-amniotic infection Hsu et al., 1998
Lyme disease Sprenger et al., 1997
Chlamydia infection Rasmussen et al., 1997
Neoplastic Squamous cell carcinoma Tettlebach et al., 1993
Melanoma Richmond et al., 1982, Pichon and Lagarde, 1989;
Rodeck et al., 1991; Luan et al., 1997
Basal cell carcinoma (sclerosing variant) Tettlebach et al., 1993
Bladder carcinoma Anisowicz et al., 1987
Colon carcinoma Cuenca et al., 1992
Other Verruca vulgaris Tettlebach et al., 1993
Keratoacanthoma Tettlebach et al., 1993
Viral (HIV) Dezube et al., 1992
Herpes simplex virus Yan et al., 1998
Cytomegalovirus Grundy et al., 1998
Rotavirus Casola et al., 1998
Reprintedwithmodificationfrompage294of‘‘HumanCytokines,HandbookforBasicandClinicalResearchIII’’
(ShattuckandRichmond,1998)withpermission.
induce concentration-dependent directional migra- in size, respectively) separated by introns of 98, 113,
tionandactivationofleukocytes.ThegenesforCXC and 531bp for the (cid:11) gene (Baker et al., 1990). The
chemokines map to human chromosome 4q13-21, sizes of exons for the (cid:12) gene are 102, 122, 84, and
with the exception of SDF-1, which is on chromo- (cid:24)700bp, respectively, while the intron sizes for the (cid:12)
some 10 (Miller and Krangel, 1992; Shirozu et al., gene are 95, 118, and 826bp, respectively (Haskill
1995; Walz et al., 1996). The family displays four etal.,1990).Forthe(cid:14) gene,therearetwoexons(178,
highly conserved cysteine residues, with the first two 123 in size) separated by two introns (97 in size for
cysteines separated by one nonconserved amino acid firstintron)(Shattuck-Brandtetal.,1997).Thesizeof
residue. The percentage identity based on nucleotide the second intron could not be determined because
sequencebetweenfamilymembersisnotstrong(43% clear intron/exon boundaries were not identified in
to 24%) (Figure 1). thesequencebeyondexon2wherethehomologyto(cid:11),
(cid:12) genes diverges. Two isoforms of CINC-2, (cid:11) and (cid:12),
Regulatory sites and corresponding are encoded by mRNAs produced by alternative
splicing. Each isoform is encoded in four exons, and
transcription factors
exon–intron boundaries are placed identically within
the aligned sequences of CXC chemokines (Shibata
The genomic structure of the MGSA/GRO(cid:11), (cid:12), (cid:13) et al., 1998).
genes has been determined after gene cloning and Deletion and mutational analysis of reporter
sequence analysis (Baker et al., 1990; Haskill et al., constructs driven by the 50 regulatory region of the
1990). There are four exons (179, 124, 84, and 716bp MGSA/GRO genes demonstrate the importance of
1026 Dingzhi Wang and Ann Richmond
Table 2 Available MGSA/GRO cDNA sequences
Species Accession no. Name GenBank locus References
Human X12510 MGSA/GRO(cid:11) HSMGSA Richmond et al., 1988
M36820 MGSA/GRO(cid:12)/MIP-2(cid:11) HUMGROB5/HSMIP2A Haskill et al., 1990;
Tekamp-Olson et al., 1990
M36821 MGSA/GRO(cid:13)/MIP-2(cid:12) HUMGROG5/HSMIP2B Haskill et al., 1990;
Tekamp-Olson et al., 1990
U88432 MGSA/GRO(cid:14) Shattuck-Brandt et al., 1997
Mouse X53798 MIP-2 MMMIP2 Tekamp-Olson et al., 1990
J04596 KC MUSSPKC Oquendo et al., 1989
Rat D11444 GRO/KC/CINC-1 RATMRSA/RA TGRO Huang et al., 1992a;
Konishi et al., 1993
D21095 CINC-2(cid:11) and (cid:12) RATCINC2 Nakagawa et al., 1994
X65647/U45965 MIP-2/CINC-3 RNMIP2/RNU45965 Driscoll et al., 1993
Hamster J03560 GRO CRUGRO Anisowicz et al., 1987
Rabbit L19157 GRO RABGRO Johnson et al., 1994
U12310 GRO homolog OCU12310 Schwartz et al., 1994
L28933 RPF2 RABRPF2X Johnson et al., 1994
Bovine O46677 GRO(cid:12) GROB_BOVIN Modi et al., 1998
O46676 GRO(cid:11) GROA_BOVIN Modi et al., 1998
O46675 GRO(cid:13) GROG_BOVIN Modi et al., 1998
Sheep U95814 GRO Modi et al., 1998
Pig U95810/U95809 GRO Modi et al., 1998
Chicken M16199 9E3/pCEF4 CHKEF9E3 Sugano et al., 1987
Reprintedwithmodificationfrompage267of‘‘HumanCytokines,HandbookforBasicandClinicalResearchIII’’
(ShattuckandRichmond,1998)withpermission.
two regions within this reporter construct which tissuesandinresponsetocertainagents(Haskilletal.,
contribute to basal promoter activity and cytokine 1990; Anisowicz et al., 1991; Cuenca et al., 1992;
inducibility (Shattuck-Brandt et al., 1994; Wood and Beckeretal.,1994).Also,the50 flankingregionofthe
Richmond, 1995). The region closest to the TATA CINC-2 contains a TATA box and putative binding
box ((cid:255)30 to (cid:255)25) contains the NF(cid:20)B consensus sites for NF(cid:20)B and AP-1 (Shibata et al., 1998).
element (at (cid:255)64to (cid:255)74 in (cid:12) and (cid:255)66 to(cid:255)76 in the (cid:11) However,nofunctional AP-1-binding sites havebeen
and (cid:13) genes) which is necessary for basal and identified in the MGSA/GRO(cid:11), (cid:12), and (cid:13) promoter
cytokine-induced transcription (Anisowicz et al., region. In addition, the 30 UTRs of the MGSA/GRO
1991). Furthermore, the HMG-I(Y) motif nested genes contain a 7bp motif (TTTTGTA) which is
within the NF(cid:20)B element is also important for basal reportedtoberequiredforactivationoftranscription
and cytokine-induced transcription (Wood et al., of the murine CC chemokine JE (Freter et al., 1992).
1995).AdjacenttotheNF(cid:20)BelementisaC/EBP-like The 7bp motif of MGSA/GRO has been demon-
element called immediate upstream regulatory (IUR) strated to contribute the stabilization of its mRNA
element. This element only contributes to the basal (Stoeckle, 1991, 1992; Stoeckle and Guan, 1993;
transcription. The SP-1 element in another region Shattuck-Brandt et al., 1994; Sirenko et al., 1997).
also contributes to the basal transcription of the The transcriptional regulation of MGSA/GRO
MGSA/GRO(cid:11)(Woodetal.,1995).Sequenceanalysis requires multiple transcription factors. The NF(cid:20)B is
of the 50 regulatory regions of each of these three essential for MGSA/GRO gene transcription
genes does reveal potential differences that might (Anisowicz et al., 1991). Melanoma cells (Hs294T)
account for selective isoform expression in certain exhibit high basal endogenous expression of the
MGSA/GRO 1027
MGSA/GRO(cid:11) gene, while the nonmalignant normal with 120 minutes in RPE cells. The more rapid
retinal pigment epithelial cells (RPE) do not exhibit turnover of I(cid:20)B(cid:11) in Hs294T as compared with RPE
basalexpression ofMGSA/GRO(cid:11), (cid:12) or (cid:13) (Shattuck- cellsresultsinaconstitutivenucleartranslocationand
Brandt et al., 1994). The constitutive MGSA/GRO activation of NF(cid:20)B, accompanied by endogenous
gene expression in the Hs294T cells is the result of transcription of MGSA/GRO in Hs294T cells.
high basal transcription of the MGSA/GRO genes. Cytokines such as IL-1 or TNF(cid:11) induce the
The high basal transcription is due to a significant expression of all three MGSA/GRO genes in RPE
increase in the binding of NF(cid:20)B p50 and p65 homo- cells (Jaffe et al., 1993). The induction of MGSA/
dimersandheterodimerstotheNF(cid:20)Belement(Wood GRO transcription by IL-1 and TNF(cid:11) was shown to
and Richmond, 1995; Wood et al., 1995). This incre- utilize the NF(cid:20)B pathway in normal human RPE
ase in NF(cid:20)B binding activity and transactivation cells, foreskin fibroblasts, and HeLa cells. However,
correlateswithalowerleveloftheinhibitorofNF(cid:20)B, cytokinesfailtoinduceMGSA/GROgeneexpression
I(cid:20)B(cid:11),andhigheractivationofIKK(cid:11)and(cid:12),compared in the Hs294T melanoma cells, although increased
with RPE cells (Shattuck-Brandt and Richmond, NF(cid:20)B p50 and p65 homodimer and heterodimer
1997; Devalaraja et al., 1999). The half-life of the complexes in the nucleus can be demonstrated by gel
I(cid:20)B(cid:11)proteininHs294Tcellsis45minutes,compared mobility shift assay. This result indicated that the
Figure 1 Coding sequence of MGSA/GRO in different species.
1028 Dingzhi Wang and Ann Richmond
Figure 1 (Continued)
MGSA/GRO 1029
Figure 1 (Continued)
regulation of transcription of the MGSA/GRO genes cell types. In the in vivo lung model, the activation
is different in the melanoma cells as compared with of NF(cid:20)B in the nuclear extracts from lung lavage
the nonmalignant control. cells correlates with the increases in CINC mRNA
The NF(cid:20)B element in MIP-2 is conserved in levels (Blackwell et al., 1994). They have shown that
sequence and location to the MGSA/GRO(cid:11), (cid:12), (cid:13) NF(cid:20)B in the lung is activated by endotoxemia with
genes. The LPS-induced transcriptional activation of consequent increased expression of CINC mRNA,
the MIP-2 gene requires an NF(cid:20)B element (Widmer secretion of biologically active CINC, and the
et al., 1993). MIP-2 is induced by TNF, LPS, and development of neutrophilic lung inflammation.
IL-1 through an NF(cid:20)B-dependent process in the
hepatic stellate cell (HSC) after a fibrogenic stimulus Cells and tissues that express
(Widmer et al., 1993; Hellerbrand et al., 1998). In
the gene
addition,MIP-2alsoexhibitshighbasaltranscription
inmurinemelanomacells(Widmeretal.,1993).These
data suggest a common mechanism for cytokine- There is endogenous expression of the MGSA/GRO
mediated regulation of the MIP-2 gene in different genes in a number of tissues including keratinocytes,
1030 Dingzhi Wang and Ann Richmond
monocytes and macrophages, mammary epithelial differences in the receptor binding specificity for
cells, and endothelial cells. However, in most tissues, IL-8 and MGSA/GRO binding to the receptor.
these genes are not expressed at detectable levels in The solution structure of murine macrophage
theabsenceofanactivatingagent,suchasacytokine, inflammatoryprotein2(MIP-2)hasbeendetermined
growth factor, endotoxin, lectin, cycloheximide, by two-dimensional homonuclear and heteronuclear
thrombin, or other stimulating agents (Haskill et al., NMR spectroscopy (Shao et al., 1998). The N- and
1990). C-terminal residues (1–8 and 70–73, respectively) are
disordered. The overall structure of the MIP-2 dimer
is similar to that reported previously for the NMR
structures of MGSA/GRO and consists of a six-
PROTEIN
stranded antiparallel (cid:12) sheet (residues 25–29, 39–44,
and 48–52) packed against two C-terminal antipar-
Accession numbers
allel (cid:11) helices. At the tertiary level, the main dif-
ferencesbetweentheMIP-2solutionstructureandthe
See Table 2. IL-8 and MGSA structures involve the N-terminal
loop between residues 9 and 23 and the loops formed
by residues 30–38 and residues 53–58. At the quater-
Sequence narylevel,thedifferencebetweenMIP-2andIL-8and
MGSA/GRO results from differing inter-helical
angles and separations.
See Figure 2.
The refined three-dimensional structure of CINC
has been determined (Hanzawa et al., 1997, 1998).
The N-terminal region containing an ELR motif is
Description of protein
disordered in solution, as in other CXC chemokines.
TheoveralldimerstructureofCINCissimilartothat
The MGSA/GRO(cid:11) protein forms dimers, tetramers, ofhumanMGSA/GRO.Themajordifferenceresides
and larger aggregates uponconcentrationthat canbe in the relative position of C-terminal (cid:11) helix with
observedaftercrosslinking(Chengetal.,1992)andin respecttothe (cid:12) sheetin thedimer. Thedistancefrom
nonreducing polyacrylamide gels (Richmond et al., (cid:11)helixtothe(cid:12) sheetiswiderinCINC (15A˚ )thanin
1986). The dimers and tetramers of MGSA/GRO are MGSA/GRO (10A˚ ) and IL-8 (11.7A˚ ). CINC exists
capable of binding to receptor (Cheng et al., 1992). mainly as a monomer at a physiological concentra-
NMR confirms that MGSA/GRO(cid:11) chemokine is a tion,similartootherproteinsbelongingtothisfamily.
dimerinsolution(Fairbrotheretal.,1993,1994;Kim
et al., 1994). The K for association is 73mM at pH
d
5.0, but the K was 7.7mM when the pH was 6.6 Discussion of crystal structure
d
(Clark-Lewis et al., 1995). The forces involved in the
stabilization of the dimer for MGSA/GRO(cid:11) appear
MIP-2 hasbeen crystallized by the Lolis group (Lolis
tobeweakerthanthoseforIL-8(Kimetal.,1994).In
et al., 1992). Preliminary crystallographic analysis
a more detailed analysis of the NMR structure of
showed that the crystals belong to space group
MGSA/GRO,thesecondarystructureoftheMGSA/
P2(1)2(1)2(1) and have unit cell dimensions of
GRO dimer was shown to be comprised of a six-
a=42.7A˚ , b=59.3A˚ , and c=100.3A˚ . The molecular
strandedantiparallel(cid:12) sheetandapairofC-terminal
massoftheproteinandvolumeoftheunitcellsuggest
(cid:11) helices (Fairbrother et al., 1994). The last four C-
that there are fourmonomers in the asymmetric unit.
terminalresiduesareprobablydisorderedinsolution.
A data set to 2.3A˚ has been collected, and the self-
Theintramoleculardisulfidelinkagesforeachsubunit
rotation function identifies the presence of a non-
arebetweenCys9andCys35aswellasbetweenCys11
crystallographic 2-fold axis. This structure can be
and Cys51. These linkages have right- and left-
accessed at www.imb-jena.de/cgi-bin/ImgLib.pl?
handed order, respectively. It is noteworthy that the
CODE=1mi2.
second histidine residue in MGSA/GRO (His34) and
IL-8(His33)exhibitverydifferentpK values,5.2and
a
3.7, respectively. It is also noted that the disorder of
Important homologies
the ELR motif is greater for IL-8 than MGSA/GRO
(Clore et al., 1989; Clore and Gronenborn, 1995).
The differences in the loops 14–19, 30–38 and the There are two MGSA/GRO homologs in mouse: KC
disorder of the N-terminus probably account for the and macrophage inflammatory protein 2 (MIP-2)
Figure 2 Amino acid sequences of the human, hamster, mouse, rat, rabbit, and chick forms of MGSA/GRO. Reprinted with
modificationfrompage270of‘‘HumanCytokines,HandbookforBasicandClinicalResearchIII’’(ShattuckandRichmond,1998)
with permission.
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1032 Dingzhi Wang and Ann Richmond
(Oquendo et al., 1989; Tekamp-Olson et al., 1990). CELLULAR SOURCES AND
KC was initially identified as a platelet-derived
TISSUE EXPRESSION
growth factor (PDGF)-inducible immediate early
response gene. The amino acid sequence of MIP-2 is
Cellular sources that produce
more closely related to human MGSA/GRO
(62%) than to the amino acid sequence for mouse
KC (59%). In rat, the MGSA/GRO gene is named MGSA/GRO proteins are produced by a variety of
cytokine-induced neutrophil chemoattractant cell types in vivo and in vitro (Table 3). This includes
(CINC). The four CINC isoforms (CINC1, 2(cid:11), 2(cid:12) not only malignant melanoma cells, but also kera-
or 3) have been purified from NRK-49F cells and tinocytes, macrophages, neutrophils, and lympho-
granulation tissue (Huang et al., 1992a; Watanabe cytes, and activated endothelial cells, fibroblasts, and
et al., 1992; Konishi et al., 1993; Zagorski and hepatocytes.
Delarco, 1993; Nakagawa et al., 1994). The amino
acid sequence of the mature CINC peptide is 91.7%
identical to KC and 69.4% identical to human Eliciting and inhibitory stimuli,
MGSA/GRO. The difference in amino acid sequence
including exogenous and
between CINC-2(cid:11) and CINC-2(cid:12) consists of only
three C-terminal residues. Rat CINC-2 and CINC-3 endogenous modulators
are 63% and 67% identical to CINC-1. There is one
MGSA/GRO chicken homolog (named 9E3 or Cytokines and growth factors upregulate the expres-
pCEF-4)clonedfromachickembryofibroblast(CEF) sion of MGSA/GRO and a number of repressors
cDNA library or from RSV-transformed fibroblasts have also been identified (Table 4). It is noted that
(Martins-Green et al., 1992). The pCEF-4 was IFN(cid:11), IFN(cid:12), and IFN(cid:13) are potent inhibitors of the
identical at the amino acid sequence level to 9E3 production of monocyte-derived IL-8, MGSA/
with only oneexception in the signal peptide (Bedard GRO(cid:11), and ENA-78. However, IFN(cid:11), IFN(cid:12), and
et al., 1987; Sugano et al., 1987). There are two in IFN(cid:13) upregulate IP-10 and MIG (non-ELR) from a
rabbit (RabGRO and RBF2) (Jose et al., 1991; variety of cells. These data suggest that interferons
Johnson et al., 1994) (Figure 2). The bovine MGSA/ may shift the biological balance toward a preponder-
GRO(cid:11), (cid:12) and (cid:13) (GROA_BOVIN, GROB_BOVIN ance of non-ELR CXC chemokines.
and GROG_BOVIN), sheep MGSA/GRO (GRO),
and pig MGSA/GRO (GRO) have also been
sequenced (Modi et al., 1998).
RECEPTOR UTILIZATION
Three human receptors and two viral receptors have
Posttranslational modifications
been cloned which bind MGSA/GRO ligands:
CXCR1, CXCR2, Duffy antigen receptor for
The mRNA for MGSA/GRO(cid:11) encodes a 107 amino chemokines (DARC), Kaposi’s sarcoma human
acid precursor that includes a 34 amino acid signal herpesvirus 8 G protein-coupled receptor (GPCR)
peptide (Anisowicz et al., 1987; Richmond et al., and the herpesvirus saimiri receptor (HSV-ECRF3).
1988). Cleavage occurs on the N-terminal side of the Two mouse receptors for KC and MIP-2 have been
ASVA sequence based upon the sequence analysis of cloned. One receptor is most closely related to the
MGSA/GRO(cid:11) protein isolated from melanoma humanCXCR2receptor.Thesecondmurinereceptor
culture medium (Richmond and Thomas, 1988). for KC and MIP-2 is mDARC. The rabbit receptor,
The mature MGSA/GRO(cid:13) protein has been purified rb F3R, has been cloned and is equivalent to the
from culture medium conditioned by osteosarcoma human CXCR1 receptor regarding ligand-binding
cells and demonstrated to have an N-terminal se- specificity. The rabbit homolog of the DARC
quence of ASVVTELRCQC, indicating a signal receptor has also been cloned recently (Peiper et al.,
peptide cleavage pattern similar to that of the (cid:11) iso- 1995).These receptorsaremembersoftheGprotein-
form (Proost et al., 1993). It has been suggested that coupled, seven transmembrane domain receptor
signalpeptideforthe(cid:12) isoformiscleavedinthesame family (Table 5). The receptor’s sequence for CINC
position (Tekamp-Olson et al., 1990; Perlman and in rat and 9E3/pCEF4 in chick has not yet been
Halvorson,1983;vonHeijne,1984,1986).Thereisno reported.
evidence to support N- or O-linked glycosylation of The binding of MGSA/GRO to the CXCR2
MGSA/GRO(cid:11) (Balentien et al., 1990). receptor effects a series of immediate and secondary