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* School of Biomedical and Chemical Sciences,
Western Australian Institute for Medical Research, and
Asthma and Allergy Research Institute and School of Medicine and Pharmacology, University of Western Australia, Crawley, Perth, Western Australia
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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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Recently, we and others have shown that allergens from house dust mite and fungi, but particularly those possessing peptidase activity, are also significant modulators of epithelial function (8, 9, 10, 11, 12). The peptidases originating from the house dust mites Dermatophagoides pteronyssinus and Dermatophagoides farinae include the group 1 cysteine proteases (13), the group 3 trypsins (14), the group 6 chymotrypsins (15), and the recently identified group 9 collagenolytic serine proteases (16). These peptides, either individually or in combination, have been shown to increase permeability of epithelial cells in vitro due to their ability to degrade the tight junction proteins ZO-1 and desmoplakin (17, 19), and stimulate airway epithelial cells directly or synergize with IL-4 and IL-13 (12) to produce cytokines such as IL-6 and IL-8. These activities may not only facilitate access of inhaled allergens to dendritic cells below the epithelial layer, but also modulate the function of immune and structural cells in the airway mucosa. With regard to more peptidase-induced cytokine release, the cellular mechanism remains unclear, although members of a recently identified G protein-coupled family of cell surface receptors, designated protease-activated receptors (PARs),3 have been implicated (10, 20, 21, 22, 23), based on biochemical similarities between mite allergens and known endogenous activators of PAR such as thrombin and trypsin. We showed that endogenous peptidases caused the release of cytokines through the activation of PAR receptors on the respiratory epithelium, and that all four members of the PAR family were expressed on respiratory epithelial cells (22). In preliminary experiments designed to investigate this possibility, we showed that activation of PAR-1, PAR-2, and PAR-4, but not PAR-3, was associated with cytokine production as well as prostanoid mediator production and combined, these data provide a robust theoretical framework upon which to investigate the role of PAR in mite allergen modulation of epithelial function. Therefore, in this manuscript, we describe studies aimed at determining whether the clinically important mite allergen, Der p 1, induced cytokine release from respiratory epithelial cells by activation of either PAR-1 or PAR-2. Our studies show for the first time that PAR-2, but not PAR-1, is involved in allergen-induced cytokine release.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Synthetic agonist peptides, as well as control PAR peptides, were synthesized with amidated C termini (purity >85%; Protein Facility, University of Western Australia, Perth, Australia). Given that the human PAR-1 agonist peptide at high concentration has been shown to activate PAR-2, the more specific frog PAR-1 agonist peptide was substituted (24). The sequences of the active and control peptides, respectively, used were: PAR-1, TFLLR-NH2 and FTLLR-NH2; PAR-2, SLIGKV-NH2 and LSIGKV-NH2 (22). Thrombin was purchased from CSL (Melbourne, Australia) and Sigma-Aldrich (St. Louis, MO). Tissue culture reagents were purchased from Life Technologies (Melbourne, Australia). General chemicals were purchased from either BDH (Kilsyth, Victoria, Australia) or Sigma-Aldrich, unless otherwise stated. The human pulmonary epithelial type II A549 cell line HeLa, COS-7, Chinese hamster ovary, and HEK cell lines were obtained from the American Type Culture Collection (Manassas, VA).
Purification of Der p 1 cysteine protease
Der p 1 was isolated from fecally enriched spent growth medium essentially as described previously (17). In brief, spent growth medium was extracted twice with 0.01 M phosphate buffer, pH 7.2, containing 0.15 M NaCl (PBS) for 2 h, and the supernatant obtained after centrifugation was applied to a monoclonal (25) anti-Der p 1 affinity chromatography column prepared using Ab kindly provided by M. Chapman (Indoor Biotechnology, Charlottesville, VA). Unbound material from the column was eluted with phosphate buffer containing 0.5 M NaCl, and bound material then was eluted after re-equilibration of the column with PBS using water adjusted to pH 11.0 with ammonia. The elute was then neutralized and applied to a soybean trypsin inhibitor affinity column to remove contaminating serine proteases (26). The unbound eluate was adjusted to pH 8.0 and applied to an anion exchange column (DEAE 52; Whatman, Maidstone, Kent, U.K.), and chromatofocusing was performed. Der p 1 elution was monitored by proteolysis using the Azocoll substrate (Calbiochem, Alexandria, Australia) in the presence and absence of 5 mM cysteine. The Der p 1-containing fractions were pooled, dialyzed against 0.05 M ammonium bicarbonate, and freeze dried. The protein content of Der p 1 was then determined spectrophotometrically.
Protease activity determinations
Trypsin activity was expressed in molar terms after determining
the percentage of active enzyme using the active site titrant,
p-nitrophenyl p'-guanidino benzoate,
as described previously (22), whereas thrombin activity
was expressed in U ml-1, as determined by
the manufacturer. However, the activity of thrombin was confirmed using
N-benzoyl-DL-arginine
p-nitroanilide, as described previously (22),
before experimentation. The thrombin supplied by CSL (0.5 nM
p-nitroaniline released min-1
mg-1) was found to be 
50-fold less active on
this basis than that supplied by Sigma-Aldrich (23.4 nM
p-nitroaniline released min-1
mg-1). Thrombin from the former source was used
in cell culture experiments, whereas that from the latter was used in
Ca2+ flux studies. The proteolytic activity of
Der p 1 was determined as described above, and percentage of
activatable enzyme was determined by titration using
n-(3-carboxyoxirane-2-carbonyl)-leucyl-amino(4-guanido)
butane E64 and expressed in molar terms using a m.w. of 25,000.
Briefly, dilutions of E64 (starting concentration, 10.5 µM) were
added to a fixed amount of activated Der p 1 in a microfuge tube in
triplicates, and Azocoll substrate was added and samples were incubated
at 37°C. ODs were then determined, and the concentration of activated
Der p 1 was calculated by plotting percentage of inhibition against E64
concentrations.
Cell culture
The A549 and HeLa cells were cultured as described previously (22, 28). For initial growth, cells were seeded into 75-cm2 tissue culture flask (Nunc, Naperville, IL) and grown to confluence in Ham’s F12 Kaighn’s Modification (Life Technologies) and DMEM, respectively, supplemented with 10% (v/v) FCS and antibiotics. For subsequent experiments, cells from the flasks were trypsinized and seeded into 24-well tissue culture plates (Nunc) at a density of 5 x 104 cells well-1 and grown to 80% confluency. At this time, the cells were then incubated in basal medium for a further 24 h. At all stages of culture, cells were maintained at 37°C in 5% (v/v) CO2.
PAR-1 and PAR-2/enhanced yellow fusion protein (EYFP) expression constructs
PAR-1 and PAR-2 plasmids in pRK7 vector constructs were obtained from L. Brass (University of Pennsylvania, Philadelphia, PA) (29) and J. Sundelin (Lund University, Lund, Sweden) (30), respectively. The plasmids were amplified using a 5' primer annealed to the Sp6 promoter 5'-GAT TTA GGT GAC ACT ATA G-3', and a 3' primer that annealed to the 3' end of the receptor sequence, and mutated out the stop codon by transforming it into a XhoI recognition site. The PAR-1 and PAR-2 primers used were 5'-CAG TCC CTT CTC GAG AGT TAAA CAG-3' and 5'-GAC CTG GAA CTC GAG ATA GGA GGT C-3', respectively. The PCR products were digested with HindIII (site already present in the pRK7) and XhoI, and cloned into pcDNA3/EYFP, as described previously (28).
Transient transfection of HeLa cells
HeLa cells were split and seeded into 100-mm tissue culture dishes (Falcon) at a cell density of 1 x 106 cells/plate, and cells were grown overnight to 60% confluence in DMEM supplemented with 10% (v/v) FCS. Transient transfections were performed the following day with 3 µg DNA (determined to be optimal by experimentation) using Superfect (Qiagen, Melbourne, Australia), as described previously (31). Cells were then split and seeded onto 12-well dishes or glass coverslips 24 h after transfection. The transfected cells were then used in assays to determine the functional significance of PAR receptors 48 h posttransfection.
Confocal imaging
HeLa cells transfected with PAR-1/EYFP and PAR–2/EYFP constructs were plated onto poly(L-lysine)-coated eight-well chamber slides 24 h after transfection, and treatment regimes were performed 48 h posttransfection. After treatment, cells were fixed in 4% (v/v) paraformaldehyde, mounted in Fluoroguard (Bio-Rad, Melbourne, Australia), and sealed with a glass coverslip. Cells were excited at 488 nm and examined using a Bio-Rad confocal laser microscope under an oil immersion objective x60 with light filtered in the green channel from 500 to 550 nm, and data were collected.
Stimulation of epithelial cells and HeLa cells
Epithelial cells were grown in appropriate serum-free basal medium for 24 h, as described previously, and exposed to varying concentrations of Der p 1 activated with 5 mM cysteine, or PAR agonist peptides. In experiments designed to study Der p 1- or PAR-induced cytokine secretion at different times during culture, cells were exposed to the optimum concentrations of Der p 1 or peptide, as described previously (22). Culture supernatants were collected, centrifuged at 12,000 x g for 5 min at 4°C, and stored frozen. At the conclusion of each experiment, cells were detached, and viability and cell number were determined by trypan blue exclusion. Epithelial cells exposed to the various additives were subsequently analyzed for potential cytotoxic reactions by measuring the release of lactate dehydrogenase using a spectrophotometric assay, as described previously (22).
Determination of IL-6 and IL-8 production
IL-6 and IL-8 production was determined using specific ELISA, as described previously (20). Briefly, 96-well plates (Maxisorp; Nunc) were coated with 100 µl/well-1 of the appropriate Ab (250 ng ml-1 in 0.1 M NaHCO3/NaCO3 buffer, pH 9.6) and incubated overnight at 4°C. The plates were then washed three times with washing buffer (PBS, pH 7.5, containing 0.5% (v/v) Tween 20) and blocked by incubating with 100 µl blocking buffer/well-1 (washing buffer containing 1% (w/v) BSA) at room temperature for 1 h. The plates were washed three times with washing buffer before 100 µl test or standards were added to the wells. Plates were incubated overnight at 4°C, washed, and incubated with biotinylated secondary Ab (100 µl 250 ng ml-1 stock in blocking buffer/well) at room temperature for 1 h. After washing, 100 µl peroxidase-labeled streptavidin (125 ng ml-1; Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added to each well, incubated at room temperature for 30 min, and, after washing, incubated with 100 µl peroxidase substrate (K-Blue ELISA substrate; Graphic Scientific, Brisbane, Australia) per well. Reactions were terminated by the addition of 100 µl 1 M phosphoric acid/well, and OD was determined using a microplate reader (Spectramax 250; Molecular Devices, Sunnyvale, CA) at 450 nm. The concentrations of cytokines in each sample were determined by interpolation from the standard curve using the SoftMax-Pro software (Molecular Devices) and expressed as pg 2.5 x 105 cells-1. Second Abs used in ELISA were obtained from BD PharMingen (San Diego, CA).
Measurement of cytosolic free calcium (Ca2+)
Changes in cytosolic Ca2+ concentration were measured using the fluorescent Ca2+ indicator, Indo-1 acetoxymethylester (AM; Teflabs, Austin, TX), as described previously (20). The ratio of fluorescence emission at 405 and 490 nm was measured using a spectrophotometer (Cairn, Faversham, Kent, U.K.) attached to an inverted microscope (Nikon, Tokyo, Japan), configured for epifluorescence. The excitation wavelength of 340 nm was provided by a variable monochromator system (Cairn). A549, transfected and nontransfected HeLa cells were grown on a coverslip to 80% confluency and loaded with Indo-1 by incubation in RPMI (medium free of phenol red) containing 6 µM membrane-permeable form of the Ca2+ indicator, Indo-1 AM, and 0.1% (w/v) Pluronic F-127 (Molecular Probes, Eugene, OR) for 45 min at room temperature. After incubation, cells were washed with two changes of RPMI to remove the excess dye and then exposed to the various test substances after establishing baseline. The ratio of the emission intensities was used as a measure of changes in cytosolic Ca2+. In experiments designed to measure receptor specificity, A549 cells loaded with Indo-1 dye were initially exposed to either peptidases or peptides and then exposed to the second stimulus after ensuring that the calcium flux reached a baseline, or after 3 min if changes in Ca2+ were not observed.
Statistical analyses
Unless stated otherwise, data are expressed as mean ± SEM. Statistical significances between means were determined using ANOVA or the Student’s t test using Microsoft Excel for Macintosh. Bonferoni’s correction was used for multiple t test comparisons.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Activated Der p 1 caused significant release of IL-6 and IL-8 from
A549 cells in a dose-dependent manner over a 24-h period (Fig. 1
, A and B) when
compared with medium controls and nonactivated allergen. The mean
maximum concentrations of IL-6 and IL-8 released in response to Der p 1
were obtained with 0.8 and 8 nM, respectively, although significant
IL-6, but not IL-8, release was observed at 0.008 and 0.08 nM (Fig. 1, A and B). Der p 1-induced IL-6 and IL-8
production was reduced at peptidase concentrations higher than those
shown to be optimal (Fig. 1, A and B). Both IL-6
and IL-8 release, in response to Der p 1 exposure, increased with
time and, for Der p 1-induced IL-6 and IL-8, maximum production was
observed at 24 h (Fig. 1, C and D), but had
not reached a plateau.
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Pretreatment of A549 cells with Der p 1 for 2 h at 37°C,
followed by exposure with either PAR-1 or PAR-2 agonist peptides at 400
µM resulted in little or no IL-6 production after PAR-1 agonist
peptide stimulation at all concentrations tested (Fig. 2A), but only significantly
reduced IL-6 production with PAR-2-stimulated cells at the 80-nM Der p
1 dose (Fig. 2B). Nonactivated Der p 1 did not cause any
significant cytokine release (Fig. 2).
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Der p 1 (80 nM) induced changes in intracellular
Ca2+ in the A549 cells (Fig. 3A) that were inhibited by
pretreatment of the enzyme with the cysteine protease inhibitor for Der
p 1, E64 (Fig. 3B). Exposure of the A549 cells to 80 nM Der
p 1 before either thrombin (Fig. 3C) or trypsin (Fig. 3D) treatment resulted in desensitization to the latter, but
not the former. Similarly, desensitization was obtained when cells were
treated with Der p 1, followed by PAR-2 agonist peptide (Fig. 3F), but not PAR-1 (Fig. 3E). Exposure of cells
to thrombin (Fig. 3G) or trypsin (Fig. 3H) before
Der p 1 resulted in desensitization to the latter only.
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To confirm that Der p 1 activated PAR-2, but not PAR-1 receptors
on the A549 cells, transfection studies were undertaken. A range of
suitable cell lines including COS-7, Chinese hamster ovary, HEK, and
HeLa cell lines was exposed to a range of Der p 1 and PAR agonist
peptides, and cytokine responses were determined. However, with the
exception of the HeLa cell line (Fig. 4A), the other cell lines
produced cytokines in response to both Der p 1 and PAR-2 agonist
peptide treatment (data not shown). HeLa cells were, therefore, used in
the transfection studies. Cells transfected with optimal concentration
of PAR-2, but not PAR-1 (data not shown) cDNA secreted significant
concentration of IL-6 in response to PAR-2 agonist peptides, and 0.4 nM
trypsin and 8 nM Der p 1 (Fig. 4B). These agonists had
little significant influence on cytokine release from HeLa cells
transfected with vector alone (Fig. 4C), although
constitutive production appeared elevated compared with nontransfected
cells. Examination of medium from cells exposed to either peptidase or
peptides for lactate dehydrogenase activity indicated that PAR
treatment did not result in cell damage (data not shown).
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Neither the PAR-2 agonist peptide nor Der p 1 induced changes in
intracellular [Ca2+] in the nontransfected or vector
alone-transfected HeLa cells, as judged by the fluorescence ratio (Fig. 5A–D). In contrast, both
stimuli caused an increase in intracellular [Ca2+] in the
PAR-2-transfected HeLa cells (Fig. 5, E and F)
within a few seconds. Changes in [Ca2+] were not observed
when HeLa cells transfected with PAR-1 plasmid were exposed to PAR-1
agonist peptide or 80 nM Der p 1 (data not shown).
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Confocal microscopy showed that HeLa cells transfected with
either PAR-1/EYFP (Fig. 6A–D) or PAR-2/EYFP cDNA
(Fig. 6E–H) expressed protein on the plasma membrane. When
the cells were exposed to 80 nM Der p 1, internalization of PAR-2, but
not PAR-1 (Fig. 6, B and C) was observed. With
regard to PAR-2, this occurred within 30 min of adding the activated
Der p 1 (Fig. 6, F and G). Nonactivated Der p 1
did not result in internalization (data not shown). After
removal of the enzyme, PAR-2 receptors reappeared on the HeLa cell
membrane within 120 min (Fig. 6H). Control experiments
performed using agonist peptide showed PAR-1 receptors were capable of
internalization, which occurred within 30 min of exposure (Fig. 6D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The involvement of PAR in these responses was investigated by pretreating A549 cells with activated enzyme before treatment with agonist peptide. We chose a 2-h pretreatment on the basis of data showing that in mouse lung, regeneration of PAR-2 receptors on the cell surface after trypsin treatment was completed within this time frame (32). The results obtained showed that Der p 1 pretreatment appear to ablate PAR-1, but not PAR-2 agonist peptide responses, suggesting cleavage of PAR-1 downstream from the thrombin cleavage site, data consistent with those derived using a range of endogenous peptidases (33, 34, 35, 36). For example, pretreatment of endothelial cells with cathepsin G, human neutrophil elastase, or proteinase 3 for 5 min has been shown to render PAR-1 refractory to subsequent activation by thrombin. However, the findings that the PAR-1 agonist peptide activated the receptor despite pre-exposure to peptidase appear to contrast with our data in which a lack of agonist peptide activation of PAR-1 was demonstrated. It is likely these differences reflect the use of the human PAR-1 agonist peptide rather than the frog agonist peptide because the human version was used at concentrations known to activate PAR-2, a receptor known to be present on endothelial cells (37).
The finding that Der p 1 pretreatment appeared to inactivate PAR-1, but activate PAR-2 formed the basis for further studies, and, to this end, both Ca2+ flux and transfection studies were used to confirm our previous observations. We showed that activated, but not E64-inhibited Der p 1 induced changes in cytosolic [Ca2+] in the A549 cell line, and that allergenic peptidase pretreatment abolished PAR-2 agonist peptide and trypsin-induced [Ca2+] flux, but not that induced by PAR-1 agonist peptide or thrombin. These findings indicate that PAR-1 remains sufficiently intact after Der p 1 treatment to respond appropriately to agonist peptide, in contrast to data obtained in the cytokine studies in which pretreatment ablated subsequent PAR-1-mediated responses. However, these differences are likely to reflect either the relatively short exposure times or Der p 1 concentrations used, or both.
Before embarking on transfection studies, we tested a number of cell lines for their suitability, but only HeLa were found to be appropriate, and these data are consistent with those reported by others (38). Transfection studies confirmed that Der p 1 activated PAR-2, but not PAR-1, as judged by both cytokine production and Ca2+ flux, and similar conclusions were drawn from studies performed with HeLa cells transfected with EYFP-tagged PAR-1 and PAR-2. We showed that Der p 1 treatment caused internalization of tagged PAR-2, but not PAR-1, and that complete internalization was obtained within 5 min of exposure. Our studies also indicated that PAR-2 receptors were re-expressed on the transfected HeLa cell surface within 2.5 h post-Der p 1 treatment. These data are similar to those obtained with green fluorescent protein-tagged PAR-2 receptor in Kirsten murine kidney epithelial cells (39), in which complete internalization of the receptor was apparent within 10 min posttrypsin exposure, and receptor re-expression was observed within 4 h of enzyme removal.
Our data suggest that Der p 1 inactivates PAR-1 by specific cleavage of
the receptor. Although detailed knowledge of the cleavage specificity
of Der p 1 is lacking, a number of susceptible residues have been
determined using synthetic peptides representing sections of occludin
(between L-L, G-T, Y-G) and claudin (between L-L, N-L, L-N, Y-G)
(18), natural protein substrates such as
1-antiprotease (between
(12D-13T,
6D-7A)
(40) and CD23 (between
298E-299S,
155S-156S)
(41), and synthetic substrates containing C-terminal R or
K (26, 42). Using this information, several possible sites
in the extracellular NH2-terminal domain, and
first and second extracellular loop regions of PAR-1 known to be
involved in the activation process can be identified (reviewed in Refs.
43 and 44) (Fig. 7). In this regard, point and deletion
mutation studies have highlighted the importance of the region
comprising residues 83Q to
89S in NH2-terminal domain
(43, 45), suggesting that Der p 1 cleavage may involve, at
the very least, 91D-92A.
The ECL1 and 2 regions of PAR-1 also contain several Der p 1 cleavable
sites, one of which namely,
258L-259N, is
absent from PAR-2 and, if cleaved in PAR-1, would result in the direct
loss of the essential 256D (43) as
well as, perhaps, altering the receptor conformation in the region of
260E, a residue also thought to be important in
the activation process (46).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Geoffrey A. Stewart, Department of Microbiology, University of Western Australia, 35 Stirling Highway, Crawley, Western Australia, 6009. E-mail address: geoffrey{at}cyllene.uwa.edu.au
3 Abbreviations used in this paper: PAR, protease-activated receptor; AM, acetoxymethylester; EYFP, enhanced yellow fusion protein.
Received for publication May 28, 2002. Accepted for publication August 12, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-arrestin-1 tagged with green fluorescent protein: -arrestin-dependent endocytosis of a proteinase receptor. J. Biol. Chem. 274:18524.1-antitrypsin by specific reactive centre loop cleavage: a mechanism that promotes airway inflammation and asthma. Biochim. Biophys. Acta 221:59.
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E. C. Cates, R. Fattouh, J. Wattie, M. D. Inman, S. Goncharova, A. J. Coyle, J.-C. Gutierrez-Ramos, and M. Jordana Intranasal Exposure of Mice to House Dust Mite Elicits Allergic Airway Inflammation via a GM-CSF-Mediated Mechanism J. Immunol., November 15, 2004; 173(10): 6384 - 6392. [Abstract] [Full Text] [PDF]
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V. M. Shpacovitch, G. Varga, A. Strey, M. Gunzer, F. Mooren, J. Buddenkotte, N. Vergnolle, C. P. Sommerhoff, S. Grabbe, V. Gerke, et al. Agonists of proteinase-activated receptor-2 modulate human neutrophil cytokine secretion, expression of cell adhesion molecules, and migration within 3-D collagen lattices J. Leukoc. Biol., August 1, 2004; 76(2): 388 - 398. [Abstract] [Full Text] [PDF]
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Y. Sakata, K. Arima, T. Takai, W. Sakurai, K. Masumoto, N. Yuyama, Y. Suminami, F. Kishi, T. Yamashita, T. Kato, et al. The Squamous Cell Carcinoma Antigen 2 Inhibits the Cysteine Proteinase Activity of a Major Mite Allergen, Der p 1 J. Biol. Chem., February 13, 2004; 279(7): 5081 - 5087. [Abstract] [Full Text] [PDF]
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 J. R. Johnson, R. E. Wiley, R. Fattouh, F. K. Swirski, B. U. Gajewska, A. J. Coyle, J.-C. Gutierrez-Ramos, R. Ellis, M. D. Inman, and M. Jordana Continuous Exposure to House Dust Mite Elicits Chronic Airway Inflammation and Structural Remodeling Am. J. Respir. Crit. Care Med., February 1, 2004; 169(3): 378 - 385. [Abstract] [Full Text] [PDF]
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) Der p 1 (8 nM). Supernatants
were collected at 3, 6, 9, 12, 18, and 24 h poststimulation, and
cytokine assays were performed. Data are expressed as above from three
independent experiments performed in quadruplicate. The concentrations
of cytokine secreted in response to Der p 1 at all time points tested
were significantly different from the medium-induced responses at the
corresponding time points, as determined using ANOVA and the Student’s
t test incorporating Bonferoni’s correction; *,
p < 0.05; **, p <
0.001.
, and
the critical residues involved in activation are marked with #. The
underlined regions indicate the residues corresponding to the agonist
peptide region (APR), and the NH2-terminal extracellular
residues (ECD) and extracellular loop residues (ECL) required for
activation (reviewed in Refs.