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Cell migration activated by platelet-derived growth factor receptor is blocked by an inverse agonist of the sphingosine 1-phosphate receptor-1

Catherine M. Waters^*, Jaclyn Long^*, Irina Gorshkova, Yuko Fujiwara, Michelle Connell^*, Kristen E. Belmonte, Gabor Tigyi, Viswanathan Natarajan, Susan Pyne^* and Nigel J. Pyne^*,1

^* Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, Glasgow, UK;
Department of Medicine, University of Chicago, Center for Integrative Science Building, Chicago, Illinois, USA;
Respiratory and Inflammation Center for Excellence in Drug Discovery, Glaxo Smith Kline, King of Prussia, Pennsylvania, USA; and
Department of Physiology, University of Tennessee Health Science Center Memphis, Memphis, Tennessee, USA

¹Correspondence: Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor St., Glasgow, G4 0NR, UK. E-mail: n.j.pyne{at}strath.ac.uk

SPECIFIC AIMS

We have previously identified a novel complex between the PDGFß receptor and the S1P₁ receptor. The complex permits the utilization of active G-protein subunits (made available by constitutively active S1P₁ receptor) by the PDGFß receptor kinase to transmit signals to p42/p44 MAPK in response to PDGF. The specific aims of the study were to 1) establish whether an inverse agonist of the S1P₁ receptor (SB649146), which functions to reduce constitutive activity of the S1P₁ receptor can be used to block PDGFß receptor signal transmission, 2) use SB649146 to define the functional role of the PDGFß receptor-S1P₁ receptor complex in regulating cell migration in response to PDGF.

PRINCIPAL FINDINGS

1. PDGFß receptor-S1P₁ receptor complexes in HEK 293 and ASM Cells
We have shown that the PDGFß receptor forms a complex with the S1P₁ receptor, which is functionally active in HEK 293 cells. The important feature of this model is that pertussis toxin (PTX), which functions to uncouple G_i from respective GPCR, reduced the PDGF-induced activation of p42/p44 MAPK. These findings are supported by data showing that the overexpression of the C-terminal tail of GRK2 (CT-GRK2), which sequesters Gß subunits, also reduced the activation of p42/p44 MAPK by PDGF or S1P. In this study, we also used airway smooth muscle (ASM) cells as these express endogenous PDGFß receptor and S1P₁ receptor, which also form a functional signaling complex.

2. Constitutively activated S1P₁ receptor enhances the PDGF-induced activation of p42/p44 MAPK
Several lines of evidence support a model in which constitutively active S1P₁ receptor can enhance signal transmission from the PDGFß receptor to p42/p44 MAPK. In this respect, overexpressed constitutively active wild-type S1P₁ receptor potentiated the PDGF-induced activation of p42/p44 MAPK in HEK 293 cells in the absence of exogenous S1P. However, the use of wild-type S1P₁ receptor in this case, does not exclude the possibility that endogenous S1P might be released from HEK 293 cells in response to PDGF. This released S1P might potentially act on the S1P₁ receptor to induce activation of p42/p44 MAPK in response to PDGF. To evaluate this possibility, we used mutant S1P₁ receptors in which the binding of S1P is completely abolished. These mutants can also be used to evaluate constitutive receptor activity. The R120A and E121A S1P₁ receptor mutants are completely defective in S1P binding, yet are able to potentiate the PDGF-stimulated activation of p42/p44 MAPK (Fig. 1 A–C).

View larger version (28K): [in this window] [in a new window]   Figure 1. The S1P1 receptor exhibits constitutive activity that enhances PDGFß receptor-dependent activation of p42/p44 MAPK. HEK 293 cells were transiently transfected with vector or plasmid constructs encoding WT S1P1, S1P1 R120A, or S1P1 E121A receptor mutants and/or PDGFß receptor. Cells were then stimulated with and without PDGF (10 ng/mL) for 10 min. The Western blots show the A) effect of WT S1P1 overexpression on PDGF-stimulated activation of p42/p44 MAPK; B) effect of S1P1 R120A, and S1P1 E121A receptor mutants on PDGF-stimulated activation of p42/p44 MAPK. Also shown are blots probed with anti-PDGFß receptor or anti-S1P1 receptor antibodies to confirm the overexpression of each protein; C) lack of effect of S1P1 R120A and S1P1 E121A receptor mutants on basal p42/p44 MAPK activation state. Blots were probed with anti-p42 MAPK antibody to confirm equal protein loading.

3. SB649146, an inverse agonist of the S1P₁ receptor, blocks PDGF-induced signaling
The fact that the S1P₁ receptor can exist in a constitutively active state to enhance PDGFß receptor signaling has allowed us to test the hypothesis that inverse agonists that reduce the amount of constitutively active S1P₁ receptors should theoretically block the stimulation of p42/p44 MAPK by PDGF. SB649146 is a novel inverse agonist of the S1P₁ receptor that stabilizes the inactive conformation of this receptor. This is based on several lines of evidence. First, SB649146 displaced the S1P₁ receptor agonist dihydrosphingosine 1-phosphate (IC₅₀=300 nM) in HEK 293 cell membranes expressing recombinant S1P₁ receptor (Fig. 2 A). Second, SB649146 reduced basal constitutive S1P₁ receptor-induced GTPS binding by approx. 70–75% (Fig. 2B ; K_i=10 nM), and antagonized S1P-induced GTPS binding (Fig. 2C ) in HEK 293 cell membranes. Third, SB649146 reduced the S1P-induced activation of p42/p44 MAPK (Fig. 2D ). Further support for a role of the S1P₁ receptor was evident from the finding that the selective S1P₁ receptor agonist SEW2781 induced an activation of p42/p44 MAPK that was reduced by pretreating cells with SB649146 (Fig. 2D ). The addition of SB649146 alone to ASM cells or HEK 293 cells (e.g., in the absence of PDGF or S1P) induced a small stimulation of the basal p42/p44 MAPK (Fig. 2D, E ), suggesting that SB649146 can also stabilize, albeit weakly, the active conformation of the S1P₁ receptor. The partial agonist effect of SB649146 on p42/p44 MAPK was blocked by pretreating ASM cells with PTX (Fig. 2D ). The stabilization of the inactive S1P₁ receptor conformation by the inverse agonist SB649146 will reduce the amount of G_i that can be activated and made available for use by the PDGFß receptor kinase. Data presented in Fig. 2D demonstrates that the treatment of ASM cells with SB649146 reduced the PDGF-induced stimulation of p42/p44 MAPK. These findings are corroborated by evidence showing that anti-sense-mediated down-regulation of the S1P₁ receptor also substantially reduced the PDGF-induced activation of p42/p44 MAPK in these cells. We also assessed the effect of SB649146 on PDGFß receptor signal transmission in HEK 293 cells. SB649146 abolished the PDGF-induced activation of p42/p44 MAPK in these cells (Fig. 2E ).

View larger version (35K): [in this window] [in a new window]   Figure 2. Effect of SB649146 on the PDGFß receptor-S1P1 receptor complex. A–C) HEK 293 cells were transfected with plasmid construct encoding S1P1 receptor. The results show the effect of SB649146 on A) [3H] dihydrosphingosine 1-phosphate binding; B) basal S1P1 receptor-induced GTPS binding; C) S1P-stimulated GTPS binding (varied S1P concentrations) (1 and 10 µM SB649146); D) ASM cells were pretreated with and without PTX (100 ng/mL) for 24 h and/or SB649146 (10 µM) for 30 min prior to stimulation of cells with S1P (1 µM, 10 min) or SEW2871 (1 µM, 10 min) or PDGF (10–30 ng/mL, 5 min). Western blots show the effect of SB649146 on PDGF-, S1P-, and SEW2871-stimulated p42/p44 MAPK activation. Also shown is the effect of PTX on the stimulation of p42/p44 MAPK by SB649146 alone; E) HEK 293 cells were transfected with plasmid construct encoding the PDGFß receptor and then pretreated with and without SB649146 (10 µM) for 30 min prior to stimulation with PDGF at the indicated concentrations for 5 min. The Western blots show the effect of SB649146 on PDGF-stimulated p42/p44 MAPK activation in HEK 293 cells ectopically expressing PDGFß receptor.

4. Specificity of SB649146 action
SB649146 was without effect on basal GTPS binding in HEK 293 cell membranes expressing recombinant S1P₂ or S1P₃ receptors suggesting that SB649146 has no inverse or partial agonist activity against these receptors and exhibits exquisite specificity for the S1P₁ receptor. Moreover, SB649146 was without effect on basal GTPS binding in membranes expressing recombinant LPA₁ receptor suggesting that SB649146 does not directly inhibit G_i, which is coupled to the LPA₁ receptor. SB649146 was without effect on the EGF-, PMA, or LPA-induced activation of p42/p44 MAPK or PDGF-induced tyrosine autophosphorylation of the PDGFß receptor.

5. Effect of SB649146 on PDGFß-S1P₁ receptor endocytosis
The pretreatment of ASM cells with SB649146 reduced PDGF-induced endocytosis of the PDGFß receptor-S1P₁ receptor complex. SB649146 also reduced the S1P-induced endocytosis of the S1P₁ receptor, thereby validating its action at S1P₁ receptors.

6. Effect of SB649146 on PDGF-stimulated cell migration
We hypothesized that the constitutive activity of the S1P₁ receptor might enhance PDGF-stimulated ASM cell migration. Evidence that supports this possibility was the demonstration that PTX or PD098059, an inhibitor of MEK1 activation and SB649146 reduced cell migration in response to PDGF.

7. Regulation of myosin light chain (MLC20) phosphorylation
To further substantiate a link between the PDGFß receptor-S1P₁ receptor complex and cell migration, we evaluated the effect of SB649146 on MLC20 phosphorylation. Our findings demonstrated that PDGF induced the phosphorylation of MLC20 and that treatment of cells with SB649146 reduced this effect.

CONCLUSIONS AND SIGNIFICANCE

This study is the first to report that a G-protein coupled receptor inverse agonist can be used to reduce growth factor-induced receptor tyrosine kinase signaling and regulation of cell migration. This not only invokes a major paradigm shift in our understanding of how growth factors function, but also necessitates a complete reevaluation of how inverse agonists of GPCR (which also act as antagonists) function. This is of immediate interest, as >70% of all known therapeutic drugs act as GPCR antagonists, suggesting that their therapeutic efficacy might be related to action of RTK-GPCR complexes rather than simply on the GPCR alone. We have reported that constitutively active S1P₁ receptor enhances PDGFß receptor signaling. The S1P₁ receptor stimulates G_i activation in the absence of S1P. Active G_i and Gß subunits made available by the constitutively active S1P₁ receptor can then enhance PDGF-induced stimulation of the p42/p44 MAPK pathway. Therefore, by blocking the constitutive activity of the S1P₁ receptor, the inverse agonist SB649146 appears to reduce availability of the G-protein subunits for use by the PDGFß receptor. In this regard, SB649146 prevents PDGF-induced endocytosis of the PDGFß receptor-S1P₁ receptor complex, which is required for stimulation of p42/p44 MAPK.

The use of S1P₁ receptor inverse agonist SB649146 has also provided new information concerning the physiological role of the PDGFß receptor-S1P₁ receptor complex in regulating cell migration. S1P itself has an inhibitory effect on migration that is possibly mediated by the S1P₂ receptor.

View larger version (28K): [in this window] [in a new window]   Figure 3. The scheme shows that inverse agonism of the S1P1 receptor in the complex with the PDGFß receptor with SB649146 disrupts functioning of the complex and prevents PDGF-induced cell migration (GRK2, G-protein coupled receptor kinase 2; PI3K, phosphoinositide 3-kinase; Gab1, GRB-2-associated binder; Grb-2, growth factor receptor binding protein 2).

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4810fje;

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This Article Full Text (PDF) All Versions of this Article: 20/3/509 05-4810fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Waters, C. M. Articles by Pyne, N. J. Search for Related Content PubMed PubMed Citation Articles by Waters, C. M. Articles by Pyne, N. J.