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Program in Molecular, Cellular, and Developmental Biology, Division of Biology, Kansas State University, Manhattan, KS 66506
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ab to CD81 (2F7) blocks T cell development at the CD4-8- stage in fetal thymic organ culture (FTOC),3 suggesting that CD81 is essential for T cell maturation (7). Subsequent to that report, three groups generated CD81-deficient mice and found that T cell development and function appears to be normal, with two interesting exceptions (8, 9, 10). First, it seems CD81-/- T cells are hypersensitive to CD3 stimulation. Second, these mice have impaired Th2 responses. Concordantly, CD81-/- mice are less sensitive to allergen-induced airway hyperreactivity (AHR) (11).
Recently we developed two new mAbs to mouse CD81 and found that CD81 is expressed on mouse thymocytes and that it is specifically up-regulated at the CD4+8+ stage (12). CD81 is then down-regulated on CD4+8- or CD4-8+ thymocytes and is low to negative on peripheral T cells but may be up-regulated upon activation. Despite these and many other functionally interesting roles for CD81, the mechanism by which CD81 (or any tetraspanin, for that matter) exerts its function is currently unknown. A principal model for tetraspanin function involves lateral associations with other cell-surface proteins (1). For example, CD81 associates with CD19, which forms a complex with CD21 (CR2) on B cells (13).
CD81 has also gained recent attention for its activity as a receptor for the hepatitis C virus envelope protein E2 (14, 15, 16, 17). Despite the clear demonstration that E2 binds CD81, it seems that additional interactions between the virus and the host cell are required for fusion and viral entry (18, 19). It is not known what other host cell proteins may facilitate hepatitis C virus entry.
To better understand the function of CD81 on T cells, we sought to
identify CD81-associated molecules. Matrix-assisted laser desorption
ionization time-of-flight (MALDI-TOF) mass spectrometry followed
by postsource-decay (PSD) analysis and National Center for
Biotechnology Information database queries were used to identify
the major CD81-associated protein here named PG regulatory-like protein
(PGRL). PGRL is a novel, 75-kDa, Ig superfamily member which is most
similar to FPRP, a protein that associates with the PGF2
receptor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All mouse cell lines were maintained in RPMI 1640 with 10% FBS (BioWhittaker, Walkersville, MD). C6VL medium was also supplemented with 50 µM of 2-ME (Sigma-Aldrich, St. Louis, MO). COS-7 was maintained in DMEM with 10% FBS (BioWhittaker). Thymocytes were isolated from the thymi of BALB/c or C57BL/6 mice of various ages.
Antibodies
Abs used include EAT1 (anti-CD81); EAT2 (anti-CD81), previously described (12); 2F7 (anti-CD81) (Southern Biotechnology Associates, Birmingham, AL); and anti-V5 Ab (Invitrogen, San Diego, CA).
Biotinylation
Indicated cell lines were washed twice in cold PBS and resuspended in 5 ml of PBS supplemented with 2 mM of Mg2+ and 2 mM of Ca2+. EZ-Link N-hydroxysuccinimide long chain biotin (Pierce Endogen, Rockford, IL) was added at 1 mg/ml PBS and incubated at room temperature for 45 min. Cells were washed four times in cold PBS and lysed as described for immunoprecipitation.
Immunoprecipitation
Indicated cell types were lysed at a concentration of 5 x 107 cells/ml on ice for 1 h in TBS with EDTA (150 mM of NaCl, 5 mM of EDTA, 20 mM of Tris (pH 7.5)) with 1% of the indicated detergent (Triton X-100, IGEPAL CA-630 (Nonidet P-40), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), or Brij-97 (Sigma-Aldrich)) supplemented with 10 U/ml aprotinin (Calbiochem, San Diego, CA), 1 mM of PMSF, and 1 µg/ml pepstatin A. Lysates were clarified by centrifugation for 15 min at 15,000 rpm before use. Lysate was precleared for 1 h at 4°C against 10 µl of protein A-agarose (Pierce Endogen) or goat anti-mouse agarose beads (Sigma-Aldrich). Anti-CD81 Abs were captured on protein A-agarose (Pierce Endogen) at 5 µg of mAb/7.5 µl of beads, while 2 µl of anti-V5 Ab was captured on 7.5 µl of goat anti-mouse agarose (Sigma-Aldrich). Ab was preincubated with the indicated beads before addition of 2 x 107 cell equivalents. Samples were rotated overnight at 4°C. Immunoprecipitated material was washed four times in lysis buffer, separated on SDS-PAGE, and transferred to polyvinylidene difluoride.
Western blot
Biotinylated material was blotted with streptavidin-HRP (1:2,500) (Southern Biotechnology Associates). EAT2 was used at 0.5 µg/ml TBST with 2% BSA, followed by goat anti-hamster HRP (Southern Biotechnology Associates) (1:5,000). Anti-V5 was used at 1:5,000, followed by goat anti-mouse HRP (1:20,000) (Southern Biotechnology Associates) and all were visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce Endogen).
Protein purification
C6VL cells (8 x 108) were lysed in 1% Brij-97. Lysate was run over a column of EAT1 Ab covalently cross-linked to protein A-agarose beads (Pierce Endogen). Bound protein was eluted in 100 mM of glycine (pH 2.8), dialyzed, concentrated, and resolved on 8–16% Tris-glycine gradient gel (Invitrogen). Proteins were visualized with Coomassie blue and p75 was excised and subjected to trypsinolysis. MALDI-TOF mass spectrometry followed by PSD analysis was used to determine the identity of p75 (J. Leszyk, University of Massachusetts Medical Center, Worcester, MA).
RT-PCR
Total RNA was isolated from indicated mouse cell lines using RNeasy Mini kit (Qiagen, Valencia, CA) and was reverse transcribed using a Reverse Transcription System (Promega, Madison, WI). PGRL was amplified from each cell line using mouse primers PGRL-3 (5'-CCAGGCGAACCCTTAGAA) and PGRL-2 (5'-CCGCATCCTCTTCATAAAG). These primers begin amplification at the second Ig domain through to the stop codon and yield a 774-bp product. Primers for CD81 (5'-ATGGGAGTGGAGGGCTGCAC and 5'-AGTACACGGAGCTGTTCCGG) were used as a positive control.
Cloning and construction of mouse PGRL constructs
IMAGE clones 330850, 2649196, and 3593592 (Research Genetics, Huntsville, AL) were obtained and independently sequenced to verify PGRL sequence. All PIg constructs are V5-tagged PGRL proteins that were amplified from mouse IMAGE clone 2649196. PIg-1 contains only the membrane proximal Ig domain (missing amino acids 1–450); PIg-2 has the two membrane proximal Ig domains (missing amino acids 1–319); and PIg-4 represents the complete PGRL construct, missing only the putative secretion tag (amino acids 1–38) and with a mutation in the stop codon. PIg-1 was amplified using forward primer PGRL-5 (5'-GGCACTGTGTACCGGGGA) and reverse primer PGRL-2 (5'-CCGCATCCTCTTCATAAAG); PIg-2 was amplified using forward primer PGRL-3 (5'-CCAGGCGAACCCTTAGAA) and PGRL-2; and PIg-4 was amplified using forward primer PGRL-1 (5'-CTTTACCGGGTGGCTGGCACC) and PGRL-2. Purified PCR products were cloned into pcDNA3.1/V5His TOPO TA (Invitrogen). Inserts were cut out of pcDNA3.1/V5His TOPO TA using PmeI and HindIII (Promega), which removes the insert along with the V5-His tag sequence. Digestion of pSecTag2 (Invitrogen) vector with PmeI and HindIII (Promega) removes the mycHis sequences. The fragments were then purified, ligated into the modified pSecTag2, and verified by sequencing.
Construction of CD81myc construct
cDNA from the mouse T cell line TK-1 was PCR-amplified with primers for mouse/human CD81 and cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen). The insert from this construct was then cut out with HindIII and XhoI (Promega) and was ligated into pCDNA3.1/mycHis and verified by sequencing.
Transfection
COS-7 and 3T3 cells were trypsinized and plated at 70–80% confluence in a T25 tissue culture flask 24 h before transfection. Lipofectamine-PLUS (Life Technologies, Gaithersburg, MD) was used for transfections according to the manufacturer’s protocol. COS-7 was transfected with 3 µg of indicated PIg plasmid and 3 µg of mouse CD81-mycHis plasmid, and 3T3 was transfected with 6 µg of PIg plasmid. Cells were cultured for 48 h at 37°C before lysis in 1 ml of 1% Brij-97 lysis solution. Immunoprecipitation and Western blot were conducted as stated above.
Northern blot
The PIg-2 construct PCR product from amplification of IMAGE clone 2649196 was used as the cDNA probe. The probe was [32P]-labeled using the Rediprime II random primer labeling system (Amersham Pharmacia Biotech, Little Chalfont, U.K.) according to the manufacturer’s instructions. The probe was hybridized to the MTN Blot according to the manufacturer’s instructions (Clontech Laboratories, Palo Alto, CA). Briefly, the denatured probe was incubated in ExpressHyb Hybridization solution at 68°C for 1 h, rinsed twice, and then washed for 30 min in 2x SSC plus 0.05% SDS at room temperature. The blot was further washed twice in 0.1x SSC plus 0.1% SDS for 40 min at 50°C before exposure for 18 h to BioMax MS film (Eastman Kodak, Rochester, NY).
Cell migration experiments
SPI-4 cells were pretreated with 1 µg of the indicated PG in 1 ml (2.8 µM) of RPMI 1640 with 10% FBS (BioWhittaker) at 37°C overnight. Pretreated cells (6 x 104) were transferred to an 8-µm pore cell culture insert (BD Biosciences, Franklin Lakes, NJ) in serum-free RPMI 1640 supplemented with indicated Abs at 1 µg/ml. After a 4-h incubation, cells that had migrated through the membrane and fallen to the bottom of the well were counted with an Eclipse TE300 microscope (Nikon, Melville, NY). Automated cell counts were done with a Chemi-Imager using AlphaEase software, both from Alpha-Innotech (San Leandro, CA), with values indicated as an average of three x10 fields.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To identify CD81-associated molecules on T cells, T cell lines
C6VL and TK-1 or thymocytes were biotinylated before lysis in different
detergents. CD81 was immunoprecipitated with the hamster anti-mouse
CD81 mAb EAT2 and associated molecules were visualized by Western blot
with streptavidin-HRP (Fig. 1
). As noted
by others, the detergent plays a critical role in determining which and
how many proteins remain associated with CD81 (20, 21). A
number of prior reports have used CHAPS lysates to demonstrate
tetraspanin associations; however, large complexes of proteins are not
dissociated by CHAPS, perhaps remaining in micelles or
detergent-resistant domains of the membrane. Notably, in Brij-97
lysates the major CD81-associated cell-surface protein on T cells has
an apparent molecular mass of 75 kDa. The association with p75 is
largely disrupted by Nonidet P-40 or Triton X-100. Consistent with
previous experience, CD81 is poorly labeled with biotin in some cell
lines such as MBL-2, PRL-9, CH27, and MS (Fig. 1C and Fig. 4A). The reason for differential reactivity of
N-hydroxysuccinimide long chain biotin with CD81 is
not known but may involve differences in CD81 conformation or
associated proteins which influence access to the labeled amine group.
It should be noted that all of the T cell lines shown in Fig. 1 express
similar levels (median fluorescence intensity 
100) of CD81 as
determined by flow cytometry. In addition to coimmunoprecipitation with
EAT2, p75 was coprecipitated with 2F7 (Fig. 1D) and with
EAT1 (not shown). Similar experiments were conducted on human T cells
(Jurkat, HSB-2, and HPB-ALL) and gave a similar result (not shown).
Anti-human CD81 (5A6) was used to immunoprecipitate biotinylated human
T cell lysates, and Western blot with streptavidin shows a band at
75 kDa.
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To determine the identity of p75, 8 x
108 C6VL cells were lysed in Brij-97, run over a
column of anti-CD81 (EAT1)-conjugated protein A-agarose beads,
eluted in 100 mM of glycine (pH 2.8), dialyzed, concentrated, and
resolved on 8–16% gradient SDS-PAGE and visualized with Coomassie
blue. The p75 band was excised and subjected to trypsinolysis followed
by MALDI-TOF mass spectrometry and PSD analysis. Protein Prospector
software (Freeware from University of California, San Francisco,
CA) was used to query various National Center for
Biotechnology Information databases to determine the identity of
p75. Four sequences derived from p75 (KDSQFSYAVFGPRV,
RVLPDELQVSAAPPGPRG, HAAYSVGWEMAPAGAPGPGRL, and
LVAQLDTEGIGSLGPGYEDRH) matched unpublished expressed sequence tags
(ESTs) from mouse which cluster under UniGene ID number Mm.29860. These
peptides are underlined in Fig. 2. The entire purification and MALDI-TOF
mass spectrometry analysis was repeated and gave the same result. The
peptide sequences were used to identify three IMAGE clones (330850,
2649196, and 3593592) which were obtained from Research Genetics and
independently sequenced. Our sequences were then assembled with 85 ESTs
using Vector NTI to determine the final mouse PGRL sequence which we
have entered in GenBank under GID#AF411055(Fig. 2). The
75-kDa protein, PGRL, is a novel Ig superfamily member possessing an
N-terminal secretion signal sequence shown in italics in Fig. 2; one
transmembrane domain, which is bracketed in Fig. 2; and four Ig
domains, which are bracketed in Fig. 3.
PGRL contains three N-linked glycosylation sites immediately following
the first conserved cysteines of the Ig-4, Ig-2, and Ig-1 domains,
which are underlined in Fig. 3. Basic local alignment search tool
analysis of the mouse PGRL sequence against the human EST
database yielded 152 ESTs which cluster under UniGene ID Hs.332012. The
human PGRL was found in the human genome database and maps to
1q23.1 (physical position 160, 663K). Interestingly, the human PGRL
gene is not annotated. The human PGRL has five exons and possesses 90%
identity with mouse PGRL at the amino acid level, excluding the
secretion tag (Fig. 2).
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Basic local alignment search tool analysis of the human
genome database and the nr GenBank database with PGRL sequence
reveals that there are three other genes closely related to PGRL. The
similarity scores of PGRL, V7/CD101, IGSF3, and FPRP create a distinct
cluster in which any of the four proteins used as a query will return
the other three with an E value
<e-70. No other known proteins,
including other Ig superfamily members, have significant similarity to
PGRL; therefore, this set of four genes may represent a small family.
The amino acid alignment of the extracellular domain of PGRL with
V7/CD101, FPRP, and IGSF3, each beginning at its N terminus, is shown
in Fig. 3
. V7/CD101, FPRP, and IGSF3 sequences contain additional Ig
domains which have been truncated for the sake of space. V7/CD101 is
expressed on T cells and was discovered as an inhibitor of T cell
activation via the TCR (22). Nothing has yet been
published regarding IGSF3. The protein most similar to PGRL is FPRP,
with 28% identity (44% similarity) to PGRL. More specifically, Ig-4
and Ig-3 of PGRL align to the outer two Ig domains of FPRP with 33%
identity (50% similarity). FPRP was discovered for its capacity to
associate with the PGF2 receptor and dampen
its affinity for PGF2 (23). FPRP
was recently identified by two groups as the major CD81/CD9-associated
protein in nonlymphoid cells (20, 21). Both of these
reports mark the band at 75 kDa as an unknown protein.
Expression and characterization of PGRL in non-T cells
PGRL association with CD81 was evaluated in the B cell lines A20,
CH27, and MS. Cells were biotinylated, lysed in Brij-97,
immunoprecipitated with EAT2, and Western blotted with streptavidin
(Fig. 4A). As described above,
CD81 is differentially labeled with biotin. Interestingly, MS is
negative for p75 and is also negative for PGRL by RT-PCR (Fig. 4C). The identity of the prominent CD81-associated protein
in MS is unknown, but its molecular mass of 116 kDa approximates
that of FPRP.
PGRL expression in nonlymphoid organs (except spleen) was evaluated by
Northern blot. Multitissue Northern blots were obtained from Clontech
Laboratories and probed with 32P-labeled PGRL
cDNA. Each lane contains 2 µg of polyA+
purified RNA, which has been confirmed for equal loading by a
consistent signal for a housekeeping gene across all lanes. As
predicted from the broad distribution of PGRL ESTs, PGRL is expressed
in many tissues but exhibits strongest expression in the brain (Fig. 4B). PGRL appears to have a single major transcript of
2.5 kb.
PGRL expression in mouse lymphocyte cell lines was evaluated by RT-PCR. All lymphoid lines tested were found to be PGRL-positive except for MS, and only a faint signal was detected in the IL-2-dependent T cell HT-2. As a positive control, primers specific for CD81 were used on the same cDNA and all lines tested were positive.
Mapping of the CD81-PGRL association
To further characterize the interaction of CD81 with PGRL we
constructed a series of PGRL expression vectors containing either the
full extracellular domain (PIg-4), or Ig-2 and Ig-1 (PIg-2), or Ig-1
domain alone (PIg-1). Each of these constructs possesses the native
PGRL transmembrane domains and the 6-aa cytoplasmic tail fused to the
V5 epitope tag. Each construct, including PIg-4, uses the IgG secretion
signal derived from the pSecTag2 vector rather than the native PGRL
secretion tag to direct the molecule to the cell membrane. Each
construct was completely sequenced and found to match the correct PGRL
sequence. To determine that the V5-tagged PGRL proteins are produced as
expected we transiently transfected 3T3 fibroblasts with each vector,
cultured 2 days, and lysed before immunoprecipitation with anti-V5
followed by anti-V5 Western blot (Fig. 5A). Each vector produced
V5-tagged PGRL of the expected sizes and in similar amounts.
Interestingly, each PGRL construct also showed a minor band of roughly
double the expected size, such as might be found with dimerization of
the PGRL-V5 product. This larger product was also seen in PIg-4 at
150 kDa but did not enter the gel shown. The small band at 35 kDa in
PIg-4 may be a degradation product.
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B, upper
panel). As a positive control, CD81 was immunoprecipitated from
the same lysates and blotted for CD81 (Fig. 5B, lower
panel).
To further confirm the interaction of CD81 and PGRL-V5, and to reduce
the possible contribution of other proteins to this interaction, we
transiently transfected COS-7 cells and conducted the same experiment.
Because COS-7 is derived from monkey, it was necessary to cotransfect
mouse CD81 in these experiments. We created a mouse CD81 expression
construct in which full-length mouse CD81 is fused to a myc
tag. This vector was completely sequenced and matched mouse CD81. As we
found in 3T3 cells, CD81 coimmunoprecipitates with PGRL containing all
four Ig domains but not with the truncated products lacking Ig-3 and
Ig-4 (Fig. 5C). The fact that this coimmunoprecipitation
works in monkey cells suggests that either the interaction is direct or
a monkey protein is able to mediate the interaction.
T cell motility is modulated by PGs and CD81
The similarity of PGRL to FPRP suggests that one natural avenue of investigation would be to study whether CD81 and/or PGRL are physically or functionally related to PG receptor activities. While the full investigation of this possibility remains the subject of a future study (and awaits production of an anti-PGRL Ab) we have found encouraging results suggesting that CD81 and PG signals may be functionally connected.
There are five classes of PG receptors, designated EP, FP, IP, TP, and
DP, and within the EP category there are four receptors that account
for the total of eight known PG receptors (24). T cells
express EP3, EP4, IP, and TP (25). The functional roles of
PG receptors on T cells are given further attention in
Discussion. Surprisingly, very little has been reported
concerning the effect of these inflammatory mediators on T cell
adhesion and motility. Both PGE1 and
PGE2 bind to EP3 and EP4 with similar affinities
(25). We tested the effect of PGE1
or PGE2 on T cell motility in the presence or
absence of anti-CD81 mAbs 2F7 or EAT1. Interestingly, EAT1 but not
2F7 in the presence of PGE1 or
PGE2 induces motility of SPI-4 mouse T cells
(Fig. 6). EAT1, 2F7, or PGs alone do not
influence T cell motility. Similar to EAT1, EAT2 also promotes T cell
motility (not shown). Exposure of T cells to PGs for 15 min or 24
h does not change the level of CD81 expression on T cells as assessed
by flow cytometry (not shown). Additionally, PG treatment does not
change the pattern of CD81-associated proteins as determined by
biotinylation-immunoprecipitation experiments (not shown).
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(not shown). None of the PGs tested
independently promotes cell-cell adhesion, nor does another Ab to mouse
CD81 (2F7) induce LFA-1-mediated adhesion in the presence or absence of
PGs (not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Interestingly, FPRP was recently identified by two groups as a major
CD81/CD9-associated protein on nonlymphoid cells. Based on their
sequence similarity and the finding that both FPRP and PGRL share an
association with CD81, it seems reasonable to consider the possibility
that PGRL may possess a function similar to FPRP.
It is worth noting that there are many parallels between the role of
PGs and the role of CD81 in T cell development and function. PGs are a
pervasive class of inflammatory mediators produced by the action of
cyclooxygenases (COX-1 or COX-2) on arachidonic acid.
PGE2 is particularly abundant in the thymus,
where its production is differentially regulated by
cortical and medullary epithelial cells and dendritic cells
(26). Mouse thymi depleted of hematopoietic cells by
2'-deoxyguanosine, such as in FTOC, produce PGE2 and
PGF2 (27). Interestingly,
COX-1-dependent synthesis of PGE2, acting via the
EP2 receptor, facilitates differentiation of T cells from the
CD4-8- stage to the
CD4+8+ stage
(28). Pharmacologic inhibition of
PGE2 synthesis impairs the adhesion of
CD4+8+ thymocytes to thymic
stromal cells, which is rescued by exogenous addition of
PGE2 (26). It is possible that these
effects are related to the fact that CD81 is specifically up-regulated
on CD4+8+ cells during T
cell differentiation and that anti-CD81 (2F7) is reported to
prevent the development of
CD4+8+ cells in
FTOC.
In the periphery, PGE2, which is produced by
macrophages, dendritic cells, and B cells, exerts a suppressive effect
on Th1 responses and augments Th2 responses. PGE2
suppresses IL-2 and IFN-
but not IL-4 or IL-5 (29).
PGE2 up-regulates macrophage-derived chemokine
production, which selectively recruits Th2 cells while suppressing
IFN-inducible protein-10, which selectively recruits Th1 cells.
Macrophage-derived chemokine production may be important for
recruitment of Th2 cells during allergic airway inflammation
(30). Additionally, PGE2 promotes
differentiation and synergistically enhances IL-4 and LPS-driven B cell
Ig class-switching to IgE (31). Interestingly,
PGD2 receptor-deficient mice exhibit greatly
reduced levels of infiltrating lymphocytes and Th2 cytokines compared
with normal mice in an allergic asthma model (32). It is
possible that there is a connection between these findings and studies
showing that CD81-/- mice have impaired Th2
responses and are resistant to AHR.
Regarding the physical nature of the PGRL-CD81 association we consider it likely that the two are directly associated. Lymphocytes generally do not express FPRP (D. Orlicky, personal communication, and our unpublished data) and therefore the association of PGRL with CD81 is not mediated by FPRP. Similarly, the mouse T cell lines used in our study do not express CD9, which excludes CD9 as a mediator of the PGRL-CD81 interaction, unlike the apparent role of CD9 as a mediator of the association between CD81 and FPRP (20). Additionally, the finding that PGRL associates with CD81 when transfected into COS-7 suggests that the interaction is direct. Future studies will determine whether a PG receptor is physically associated with PGRL and/or CD81. It will also be important to determine whether PGRL and/or CD81 influence the specific binding of a PG or the activities of a PG receptor. The specific PG receptor remains to be established, but the antagonistic effect of PGE1 and PGE2 on CD81-induced LFA-1 activation and the ability of PGE2 to facilitate T cell motility in the presence of anti-CD81 hint that PGRL may regulate PGE2 responses. This hypothesis is particularly attractive given the parallels of PGE2 and CD81 in AHR and T cell development.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Scott C. Todd, Division of Biology, Kansas State University, 18 Ackert Hall, Manhattan, KS 66506. E-mail address: stodd{at}ksu.edu
3 Abbreviations used in this paper: FTOC, fetal thymic organ culture; PGRL, PG regulatory-like protein; AHR, airway hyperreactivity; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; PSD, postsource-decay; EST, expressed sequence tag; FPRP, PGF2 receptor-associated protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; h, human.
Received for publication July 5, 2001. Accepted for publication August 22, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor associated protein (FPRP). Prostaglandins Leukotrienes Essent. Fatty Acids 54:247.[Medline]
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 C. Penha-Goncalves, C. Moule, L. J. Smink, J. Howson, S. Gregory, J. Rogers, P. A. Lyons, J. J. Suttie, C. J. Lord, L. B. Peterson, et al. Identification of a Structurally Distinct CD101 Molecule Encoded in the 950-kb Idd10 Region of NOD Mice Diabetes, June 1, 2003; 52(6): 1551 - 1556. [Abstract] [Full Text] [PDF]
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 X. A. Zhang, W. S. Lane, S. Charrin, E. Rubinstein, and L. Liu EWI2/PGRL Associates with the Metastasis Suppressor KAI1/CD82 and Inhibits the Migration of Prostate Cancer Cells Cancer Res., May 15, 2003; 63(10): 2665 - 2674. [Abstract] [Full Text] [PDF]
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 B. Fritzsching, B. Schwer, J. Kartenbeck, A. Pedal, V. Horejsi, and M. Ott Release and Intercellular Transfer of Cell Surface CD81 Via Microparticles J. Immunol., November 15, 2002; 169(10): 5531 - 5537. [Abstract] [Full Text] [PDF]
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M. E. Hemler Specific tetraspanin functions J. Cell Biol., December 24, 2001; 155(7): 1103 - 1108. [Abstract] [Full Text] [PDF]
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