| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 279, Issue 53, 55419-55424, December 31, 2004
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
-Arrestin-1 and -2*
**
From the
Division of Biomedical Sciences, **Biochemical and Molecular Biology Program and Cellular, Molecular and Developmental Biology Program, University of California, Riverside, California 92521 and the ¶Howard Hughes Medical Institute, Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
Received for publication, September 8, 2004 , and in revised form, October 12, 2004.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
-arrestin-1 reduces PAR-2-stimulated internalization, ERK1/2 activation, and cell migration; however, this reagent may block association of many proteins, including -arrestin-2 with clathrin-coated pits. Here we investigate the role of PAR-2 in the constitutive migration of a metastatic breast cancer cell line, MDA MB-231, and use small interfering RNA to determine the contribution of each -arrestin to this process. We demonstrate that a trypsin-like protease secreted from MDA MB-231 cells can promote cell migration through autocrine activation of PAR-2 and this correlates with constitutive localization of PAR-2, -arrestin-2, and activated ERK1/2 to pseudopodia. Addition of MEK-1 inhibitors, trypsin inhibitors, a scrambled PAR-2 peptide, and silencing of -arrestins with small interfering RNA also reduce base-line migration of MDA MB-231 cells. In contrast, a less metastatic PAR-2 expressing breast cancer cell line does not exhibit constitutive migration, pseudopodia formation, or trypsin secretion; in these cells PAR-2 is more uniformly distributed around the cell periphery. These data demonstrate a requirement for both -arrestins in PAR-2-mediated motility and suggest that autocrine activation of PAR-2 by secreted proteases may contribute to the migration of metastatic tumor cells through -arrestin-dependent ERK1/2 activation. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
-arrestin-1 that encodes the clathrin binding domain (12). However, the specificity of this reagent is unclear as it acts by binding -arrestin contact sites on clathrin and may affect other clathrin-regulated processes as well. In recent studies, we demonstrated that PAR-2 promotes chemotaxis by a mechanism involving localization of activated ERK1/2 and its upstream regulatory kinases to the pseudopodia. This pseudopodial localization appears to occur through the formation of a scaffolding complex containing -arrestin(s) that we refer to as an endosomal scaffold (12, 13).
While -arrestins were originally thought to mediate signal termination, there has been a growing body of evidence that they can also serve as signaling scaffolds for a number of receptors (14–16) and that further functional differences exist between the two -arrestin family members. Recent studies using siRNA to specifically knockdown either -arrestin-1 or 2 suggest that 2AR primarily utilizes -arrestin-2 for internalization, while the angiotensin II type 1a receptor (AT1aR) can use either for internalization but requires -arrestin-2 for signaling to ERK1/2 (17). Additional differences may exist between other cell types and receptors. The role of -arrestins in cell migration is supported by genetic studies demonstrating impaired CXCR-mediated motility in lymphocytes from -arrestin-2 knock-out mice (18). However, the specific -arrestin family member(s) that mediate(s) PAR-2-promoted cell motility has not been investigated nor has the role of -arrestins in tumor cell migration. The purpose of these studies was 2-fold. First, using siRNA to specifically knockdown each -arrestin, we wished to determine which -arrestins mediate ERK1/2 activation and chemotaxis downstream of PAR-2. Second, using the metastatic breast cancer cells (MDA MB-231) that migrate constitutively and cells with low metastatic potential (MDA MB-468) (19, 20) that do not migrate without stimulation, we wished to investigate how PAR-2 and -arrestins might modulate cell migration in a metastatic tumor cell.
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
-arrestin-1 (Pharmingen); mouse anti-PAR-2 Sam11 (Zymed Laboratories Inc.); rabbit anti-PAR-2 B5 (Dr. Morley Hollenberg, University of Calgary); rabbit -arrestin-1 and -2 were generated in the Lefkowitz laboratory; rabbit anti-ERK1 antibody (Santa Cruz Biotechnology). Anti-phospho-p42/44 MAPK (pERK) and MEK1 inhibitor PD98059 were from Cell Signaling Inc. Antibody dilutions for Western blotting were as follows: pERK (1:2000), ERK1/2 (1:2000), PAR-2 (SAM11, 1:250), -arrestin-1/2 (1: 100), phospho-Raf (1:1000), total Raf (1:1000), total ERK1 (1:1000), and histone (1:250). Cell Culture—MDA MB-231 and MDA MB-468 cells were grown in Leibowitz's L15 medium, supplemented with 14 mM NaHCO3 and 10% fetal calf serum and maintained at 37 °C, 5% CO2.
Microscopy—Cells were seeded onto collagen-coated coverslips for 6 h and serum-starved overnight. Protease inhibitor mixture (PIC: 100 µM leupeptin, 2 µg/ml aprotinin, 6.25 µg/ml 
1-antitrypsin, and 0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF)) or hAP were added for 5–90 min as described, and cells were then fixed in normal buffered formalin followed by pressure cooker antigen retrieval in 1% sodium citrate and stained with anti-B5 (1:500) overnight and Alexa-595 conjugated secondary. Serial sections (1 µm, 40x and 100x objectives) were taken on a Zeiss LSM510 microscope. For images in Fig. 2C, images were taken with a 2.5x zoom. For video microscopy, MDA MB231 cells were observed by phase contrast microscopy in bicarbonate-free medium on a Nikkon Inverted microscope (40x objective), and time-lapse images were taken every 2 min using Metamorph software. A quicktime video with 1.0-s delays (2 s delays indicate addition of PIC and AP) is included in the supplemental material.
|
-arrestin-1, 5'-AAAGCCUUCUGCGCGGAGAAU-3'; -arrestin-2, 5'-AAGGACCGCAAAGUGUUUGUG-3'; -arrestin-1 and -2, 5'-ACCTGCGCCTTCCGCTATG-3'; control (non-targeting sequence), 5'-AAUUCUCCGAACGUGUCACGU-3') with 19-nucleotide duplex RNA and 2-nucleotide 3'-dT overhangs were purchased from Xeragon (Germantown, MD) in deprotected and desalted form. Cells (40–50% confluent) were transfected with 20 µg/10-cm dish of siRNA, using Genesilencer. For pERK studies, cells were seeded into 6-well dishes 48 h after siRNA transfection and serum-starved overnight prior to stimulation. For chemotaxis assays, cells were serum-starved for 16 h and split into trans-well chambers 48 h after siRNA transfection. Chemotaxis Assays and Pseudopodia Purification—For all migration and pseudopodia purification assays, cells were serum-starved overnight in 0.1% bovine serum albumin then seeded onto collagen (10 µg/ml)-coated 12-mm Transwell filters with 8-µm pores for migration (104/filter) and 3-µm pores for pseudopodia purification (105/filter). Agonists (trypsin, AP, or 2f-AP) were added to the bottom chamber, and migrated cells or pseudopodia and cell bodies were analyzed as described previously (13). For inhibitor experiments, a mixture of serine protease inhibitors (PIC), 1 µg/ml soybean trypsin inhibitor (SBTI), 2 µg/ml leupeptin or 2 µg/ml aprotinin, 0–100 µM 2-furoyl-ILRGL-O, or 10 µM PD98059 was added to the top chamber after cells attached for 2 h and migration monitored as described previously.
Subcellular Fractionation—5 x 105 cells/60-mm dish (grown 24 h) were serum-starved for 16 h, treated with 50 µM AP for 0–60 min at 37 °C, washed twice, lysed by Dounce homogenization (10 strokes) in 0.25 ml of hypotonic lysis buffer, and fractionated as described previously (13). Quantification of band densities was performed using Adobe Photoshop, and pERK band densities were normalized to total ERK band densities for each lane.
Trypsin Secretion—At the start of the experiment 3 ml of fresh medium was added to cells (80% confluent) and samples were collected for 5–90 min. Protein was concentrated by chloroform/MeOH extraction and analyzed by SDS-PAGE followed by immunoblotting with antitrypsin/trypsinogen. To estimate quantities of trypsin(ogen), 1 µg of purified trypsin was included, and band densities were compared with purified trypsin using Adobe Photoshop histogram analysis.
Statistics—All graphs and statistical analyses were performed using Kaleidagraph Version 3.5.1 or Graphpad Prism, Version 3.5. Migration assays and phospho-ERK analyses were performed a minimum of three times. Analysis of variance and Tukey t tests were used to determine statistical significance and significant differences of values.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
|
Previous studies showed that MEK1 inhibitors and expression of a dominant negative ERK2 inhibit PAR-2-stimulated migration in NIH3T3 and MDA MB-468 cells (12, 13). Addition of PD98059 reduced base-line MDA MB-231 cell migration by 68 ± 3% (Fig. 1C, cross-hatched bar), suggesting PAR-2 was acting through ERK1/2 to promote migration. Consistent with this effect, base-line ERK1/2 activation was elevated in plasma membrane fractions from MDA MB-231 cells, compared with MDA MB-468 cells, and addition of hAP increased ERK1/2 phosphorylation in both MDA MB-231 and MDA MB-468 cells (Fig. 2).
MDA MB-231 Cells Secrete Biologically Active Trypsin—The ability of trypsin inhibitors to block MDA MB-231 cell migration suggests they might secrete trypsin, leading to autocrine activation of PAR-2. We compared the trypsin secreted from MDA MB-231 and MDA MB-468 cells by Western blot of conditioned medium (CM), sampled over 3 h. Trypsin appeared in the medium of MDA MB-231 cells almost immediately and continued to accumulate, while no detectable trypsin appeared in the medium of MDA MB-468 cells (Fig. 3A). The approximate concentration of trypsin of conditioned medium was calculated by comparing the density of antitrypsin immunoreactive bands to a purified trypsin control and was estimated to be in the low nanomolar range (
1–5 nM). We then tested the ability of CM from MDA MB-231 cells to or MDA MB-468 cells to promote chemotaxis of MDA MB-468 cells. While MDA MB-231 CM resulted in a 12.5 ± 1.4-fold increase in cell migration, MDA MB-468 CM had no effect. Incubation of CM with PIC abolished cell migration demonstrating that the major chemotactic factor present in the medium is a serine protease. Purified trypsin induced a similar (14 ± 2-fold) increase in cell migration (Fig. 3B).
|
|
Immunofluorescence reveals that PAR-2 localization in MDA MB-231 cells appears to be enriched at the leading edge, while in the non-migratory MDA MB-468 cells it is distributed more uniformly around the cell surface (Fig. 4B). Furthermore, treatment of MDA MB-231 cells with trypsin inhibitors results in redistribution of PAR-2 around the cell periphery, while treatment of MDA MB-468 cells with hAP results in accumulation of surface PAR-2 at membrane ruffles (Fig. 4C). In MDA MB231 cells, a considerable amount of PAR-2 is localized to intracellular compartments, probably both the Golgi apparatus, as has been reported by several other investigators (22), and the lysosomes, due to constitutive receptor activation. Of most interest to us from the perspective of cell migration is that fraction localized to the leading edge.
To determine whether PAR-2 is constitutively sequestered in extending pseudopodia along with other components of the previously described endosomal scaffold (12, 13), we purified pseudopodia and cell bodies from unstimulated MDA MB-231 cells as described previously (13, 23). Only MDA MB-231 cells were included because pseudopodia formed in response to agonist in MDA MB-468 cells are smaller and result in too little protein for analysis. In untreated MDA MB-231 cells, 95 ± 5% -arrestin-2, 65 ± 20% -arrestin-1, 75 ± 5% pERK, and 65 ± 5% pRaf are localized to the pseudopodia (Fig. 5). Total ERK1/2 and Raf-1 are distributed approximately equally between pseudopodia and cell bodies, suggesting the phosphorylated forms are specifically restrained at the leading edge. A study by Brahmbhatt and Klemke (24) comparing found that phospho-ERK was specifically associated with extending but not retracting pseudopodia, consistent with the idea that sequestration of signaling molecules to the leading edge might promote persistent migration. Interestingly, the modified forms of PAR-2 are differentially distributed. While 80% of the glycosylated PAR-2 is found in the pseudopodia, 97% of the phosphorylated protein and 70% of the unglycosylated protein are restricted to the cell body (Fig. 5). Further studies are required to determine whether glycosylation and phosphorylation play a role in localization of PAR-2 to the pseudopodia and subsequent cell migration. These data are consistent with the hypothesis that trypsin secreted from MDA MB-231 cells activates PAR-2 resulting in its incorporation into a signaling complex at the leading edge of the cell.
|
-Arrestin-1 and -2—Our previous data showed that blocking receptor internalization with a dominant negative fragment of -arrestin-1 inhibits PAR-2-stimulated ERK1/2 activation and motility (12, 13). Recent evidence suggests that -arrestins are not merely redundant family members, and while some receptors appear to depend predominantly on one -arrestin for both signaling and signal termination functions, others use one for internalization and another for signaling. It is unclear which one -arrestin PAR-2 "prefers," and the dominant negative -arrestin-1 used previously can inhibit both and may even have a more global effect on membrane trafficking. We used siRNA to knockdown -arrestin-1 and -2 expression either individually or together and found that inhibiting expression of either of the -arrestin-1 or -2 reduced base-line MDA MB-231 cell migration (by 55 ± 10% and 50 ± 12%, respectively), while simultaneous knockdown of both -arrestins resulted in a 71 ± 4% reduction (Fig. 6B). To investigate whether the effects of PAR-2 and -arrestins on cell migration was through the same pathway, we examined the effect of siRNA knockdown on PAR-2-stimulated cell motility, in the presence of PIC. As described in the legend to Fig. 1C, addition of PIC depresses base-line cell migration, and addition of hAP or 2f-AP restores migration. In the presence of PIC, siRNA knockdown of -arrestin-1 expression abolishes 2f-AP-stimulated cell migration, and knockdown of -arrestin-2 reduces 2f-AP-stimulated migration by 92 ± 6%, suggesting that PAR-2-stimulated migration requires both -arrestin-1 and -2 (Fig. 6C) and introducing the question of whether they each have distinct roles in PAR-2 signaling.
|
-arrestins in cell migration was ERK1/2 activation, we examined the effect -arrestin-1 and -2 siRNA on ERK1/2 activation after pretreatment of cells with PIC to suppress base-line PAR-2 activity. Knockdown of either -arrestin-1 or -2 expression PAR-2 stimulated ERK1/2 phosphorylation by 80–90% (Fig. 6D), suggesting both -arrestins contribute to PAR-2-stimulated ERK1/2 activation and that the distinct roles for -arrestin-1 and -2 in cell migration might lie downstream of ERK1/2. |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
-arrestin scaffolding of signaling molecules.
A specific requirement for -arrestin-2 in lymphocyte motility has recently been demonstrated using -arrestin-1 and -2 knock-out mice (18). An interesting outcome of the studies described here is the evidence that both -arrestins are required for PAR-2-mediated motility, suggesting they are not redundant, even for receptors that appear to utilize both. Such a finding is consistent with studies by others demonstrating that the two -arrestin proteins differ in their specific cellular functions (17, 18, 25–27), and their precise roles in the regulation of actin assembly machinery are described in studies from our laboratory currently in preparation.3 Previous studies in our laboratory demonstrated that migration is dependent upon -arrestins and ERK1/2 and appears to involve trafficking of the activated kinases to the pseudopodia on early endosomal vesicles (12, 13). Here we show that the components of this endosomal scaffold are constitutively localized to pseudopodia in MDA MB-231 cells. Interestingly, although both -arrestin-1 and -2 are required for cell migration, -arrestin-2 is enriched to a greater extent in the pseudopodia, consistent with the hypothesis that the two -arrestins may have separate functions in cell motility.
Studies comparing a variety of breast cancer cell lines have demonstrated that MDA MB-468 cells exhibit low levels of endothelial cell invasion, compared with MDA MB-231 cells, and are not metastatic in a nude mouse model (19, 20, 28). The fact that less metastatic tumor cells both express PAR-2 and migrate in response to its activation is interesting because a number of proteases released during inflammation can activate PAR-2 (8, 29), raising the possibility that chronic inflammation in a cancerous tissue might increase the metastatic potential of a tumor.
Most chemotherapeutic agents target cell division; because metastasis involves proliferation of tumor cells after they have invaded the blood vessel wall, the tumor cells are thought to prevent metastasis as well. However, tumor cells may possess multiple mechanisms for cell migration, which they can utilize independent of cell division. Further studies are required to determine whether trypsin secretion and PAR-2 endosomal scaffold formation are altered in a broad spectrum of tumor cells, whether PAR-2 is required for metastasis of certain tumors in vivo, the efficacy of serine protease inhibitors and PAR-2 antagonists in preventing metastasis, and the role of -arrestins in this process. The use of serine protease inhibitors as therapeutics is not a new concept, and trypsin and tryptase inhibitors have been used in the treatment of diseases such as asthma, emphysema, and 1-antitrypsin deficiency (25, 30–32). Over the last few years, however, the utility of targeting serine proteases as a means of preventing tumor metastasis has been an active area of research. While the major focus has been on the role of proteases in degrading the tumor stroma and basement membrane, the studies described here suggest that activation of cell migration pathways through cell surface receptors may also contribute to the prometastatic action of serine proteases.
|
FOOTNOTES |
|---|
The on-line version of this article (available at http://www.jbc.org) contains supplemental video 1.
These authors contributed equally to this work.
|| A Howard Hughes Medical Institute Investigator.
To whom all correspondence should be addressed: B605 Statistics Rd., University of California, Riverside, CA 92521. Tel.: 951-827-2871; E-mail: katie.defea{at}ucr.edu.
1 The abbreviations used are: PAR-2, protease-activated receptor-2; ERK1/2, extracellular signal-regulated kinase-2; AP, activating peptide; hAP, human AP (SLIGKV-NH2); 2f-AP, 2-furoyl-LIGRL-ornithine-NH2; scr-AP, scrambled PAR-2 peptide; SBTI, soybean trypsin inhibitor; PIC, protease inhibitor mixture; CM, conditioned medium; ERK, extracellular signal-regulated kinase; siRNA, small interfering RNA.
2 M. Mathur and K. DeFea, unpublished observations.
3 L. Ge and K. DeFea, manuscript in preparation.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  B. Lagane, K. Y. C. Chow, K. Balabanian, A. Levoye, J. Harriague, T. Planchenault, F. Baleux, N. Gunera-Saad, F. Arenzana-Seisdedos, and F. Bachelerie CXCR4 dimerization and {beta}-arrestin-mediated signaling account for the enhanced chemotaxis to CXCL12 in WHIM syndrome Blood, July 1, 2008; 112(1): 34 - 44. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. Ramsay, Y. Dong, M. L. Hunt, M. Linn, H. Samaratunga, J. A. Clements, and J. D. Hooper Kallikrein-related Peptidase 4 (KLK4) Initiates Intracellular Signaling via Protease-activated Receptors (PARs): KLK4 AND PAR-2 ARE CO-EXPRESSED DURING PROSTATE CANCER PROGRESSION J. Biol. Chem., May 2, 2008; 283(18): 12293 - 12304. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. H. Versteeg, F. Schaffner, M. Kerver, H. H. Petersen, J. Ahamed, B. Felding-Habermann, Y. Takada, B. M. Mueller, and W. Ruf Inhibition of tissue factor signaling suppresses tumor growth Blood, January 1, 2008; 111(1): 190 - 199. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Bouschet, S. Martin, V. Kanamarlapudi, S. Mundell, and J. M. Henley The calcium-sensing receptor changes cell shape via a beta-arrestin-1 ARNO ARF6 ELMO protein network J. Cell Sci., August 1, 2007; 120(15): 2489 - 2497. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Zoudilova, P. Kumar, L. Ge, P. Wang, G. M. Bokoch, and K. A. DeFea beta-Arrestin-dependent Regulation of the Cofilin Pathway Downstream of Protease-activated Receptor-2 J. Biol. Chem., July 13, 2007; 282(28): 20634 - 20646. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Kumar, C. S. Lau, M. Mathur, P. Wang, and K. A. DeFea Differential effects of beta-arrestins on the internalization, desensitization and ERK1/2 activation downstream of protease activated receptor-2 Am J Physiol Cell Physiol, July 1, 2007; 293(1): C346 - C357. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. R. Hamilton, S. F. Fard, F. F. Paiwand, C. Tolg, M. Veiseh, C. Wang, J. B. McCarthy, M. J. Bissell, J. Koropatnick, and E. A. Turley The Hyaluronan Receptors CD44 and Rhamm (CD168) Form Complexes with ERK1,2 That Sustain High Basal Motility in Breast Cancer Cells J. Biol. Chem., June 1, 2007; 282(22): 16667 - 16680. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Arora, T. K. Ricks, and J. Trejo Protease-activated receptor signalling, endocytic sorting and dysregulation in cancer J. Cell Sci., March 15, 2007; 120(6): 921 - 928. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Vroon, C. J. Heijnen, and A. Kavelaars GRKs and arrestins: regulators of migration and inflammation J. Leukoc. Biol., December 1, 2006; 80(6): 1214 - 1221. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Jafri, H. M. El-Shewy, M.-H. Lee, M. Kelly, D. K. Luttrell, and L. M. Luttrell Constitutive ERK1/2 Activation by a Chimeric Neurokinin 1 Receptor-beta-Arrestin1 Fusion Protein: PROBING THE COMPOSITION AND FUNCTION OF THE G PROTEIN-COUPLED RECEPTOR "SIGNALSOME" J. Biol. Chem., July 14, 2006; 281(28): 19346 - 19357. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. G. H. Scott, V. Pierotti, H. Storez, E. Lindberg, A. Thuret, O. Muntaner, C. Labbe-Jullie, J. A. Pitcher, and S. Marullo Cooperative Regulation of Extracellular Signal-Regulated Kinase Activation and Cell Shape Change by Filamin A and {beta}-Arrestins. Mol. Cell. Biol., May 1, 2006; 26(9): 3432 - 3445. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Hunyady and K. J. Catt Pleiotropic AT1 Receptor Signaling Pathways Mediating Physiological and Pathogenic Actions of Angiotensin II Mol. Endocrinol., May 1, 2006; 20(5): 953 - 970. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. K. Shenoy, M. T. Drake, C. D. Nelson, D. A. Houtz, K. Xiao, S. Madabushi, E. Reiter, R. T. Premont, O. Lichtarge, and R. J. Lefkowitz beta-Arrestin-dependent, G Protein-independent ERK1/2 Activation by the beta2 Adrenergic Receptor J. Biol. Chem., January 13, 2006; 281(2): 1261 - 1273. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. R. Morris, Y. Ding, T. K. Ricks, A. Gullapalli, B. L. Wolfe, and J. Trejo Protease-Activated Receptor-2 Is Essential for Factor VIIa and Xa-Induced Signaling, Migration, and Invasion of Breast Cancer Cells Cancer Res., January 1, 2006; 66(1): 307 - 314. [Abstract] [Full Text] [PDF]
|
= high molecular weight form. pRaf and pERK1/2 levels (P) were compared with total ERK1/2 and Raf levels (T). For PAR-2, pseudopodial/cell body ratio was calculated for the sum of all four bands (T) and for each individual band.