Transactivation of the EGF receptor

Activation of GPCRs can lead to the stimulation of RTKs, such as the epidermal growth factor receptors (EGFRs) (278), which thus creates docking sites for proteins that contain phosphotyrosine-binding domains and leads to the assembly of complexes that contain Sos (which in turn activates Ras and the MAPK pathway) (see Figure 10). Several mechanisms have been proposed to mediate the transactivation of RTKs by GPCRs, including the activation of RTKs through NRTKs°, the formation of complexes between GPCRs and RTKs°, and the release of RTK ligands. Indeed, the activation of NRTK can lead to the phosphorylation of key tyrosine residues of EGFRs (273), whereas the formation of stable molecular complexes between

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activated -adrenoreceptors and EGFRs have been demonstrated following their co-internalization into clathrin-coated vesicles (279). Furthermore, GPCRs can provoke the proteolytic cleavage and release of membrane-bound pro-hormones, such as heparin-binding epidermal growth factor (HB-EGF), by a metalloprotease that initiates an autocrine-paracrine mechanism of activation of EGFRs (see Figure12) (280).

Concerning PTH1R, transactivation of EGFR was shown to be required for ERK activation by PTH in murine osteoblasts (210). In this case, upon PTH1R stimulation, a matrix metalloprotease is activated and induces the release of an EGF ligand which in turn binds to the EGFR and induces its activation. This transactivation of the EGFR was confirmed in a study using distal kidney cells (281) and also in another study using HEK293 cells transiently expressing PTH1R, in which case transactivation occurred through a -arrestin-dependent mechanism (223). Altogether, these data lead to the hypothesis that transactivation of the EGFR induced by PTH could be implicated in PTH mitogenic effects.

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Figure 10. Multiple pathways link GPCRs (as PTH1R) to mitogen-activated protein kinase (MAPK). See text for more details. Biochemical routes initiated by -subunits can stimulate Ras by the activation of receptor tyrosine kinase (through proteolytic release of EGF ligand) and non-receptor tyrosine kinases, which results in the recruitment of Sos to the membrane and the exchange of GDP for GTP bound to Ras.

Activated Gq can stimulate Raf1 through protein kinase C (PKC) or by stimulating Ras by the Ca²⁺-dependent activation of RasGRF and tyrosine kinases acting on Sos.

Gi, Go and Gs can also use tissue-restricted pathways regulating Rap, which can stimulate B-Raf and lead to the activation of MAPK. Arrows represent positive stimulation and broken lines represent inhibition. Abbreviations: EPAC, exchange protein activated by cAMP; GAP, GTPase-activating protein; GRF, guanine-nucleotide releasing factor; MEK, MAPK kinase; PI3K, phosphoinositide 3-kinase;

PKA, protein kinase A; PLC, phospholipase C; RTK, receptor tyrosine kinase.

Adapted from Marinissen M.J., TRENDS in Pharmacological Sciences (2001).

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Studies

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RATIONALE AND HYPOTHESIS OF STUDY 1

PTH1R is a GPCR coupled to the classical Gs/cAMP/PKA and Gq/IP3/PKC signaling pathways. PTH1R stimulation by PTH and PTHrP, also leads to activation of the ERK1/2 MAPKs. A number of transduction pathways has been proposed for PTH1R-mediated activation of ERK1/2, involving cAMP/PKA, G subunit and/or PKC, -arrestin and EGF-receptor transactivation (160, 210, 223, 245, 251). The intracellular juxta-membrane region of PTH1R could be implicated in ERK1/2 activation by binding G subunits, however the PTH1R structural determinants required for ERK1/2 activation have not been extensively investigated. It is equally unknown whether c-Src may directly interact with -arrestin and/or PTH1R itself to activate ERK1/2 To clarify the molecular mechanisms of ERK1/2 activation by PTH, we performed structure-function studies of PTH1R and its downstream effectors leading to ERK1/2 activation and transcriptional activity.

AIM FOR STUDY 1

To further delineate the PTH1R structural determinants and the role of -arrestin and c-Src in ERK1/2 activation

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Study 1

Proline-rich motifs in the parathyroid hormone (PTH)/PTH-related protein receptor C terminus mediate scaffolding of c-Src with -arrestin2

for ERK1/2 activation.

Rey A., Manen D., Rizzoli R., Caverzasio J., Ferrari SL.

(2006) J. Biol. Chem 281(50), 38181-8

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SUMMARY OF EXPERIMENTAL APPROACH AND RESULTS

For this purpose we used site-directed mutagenesis of the receptor in a step-by-step approach to delineate PTH1R regions implicated in ERK1/2 activation. More precisely, we mutated various motifs in the PTH1R C terminus and the second intracellular loop (IC2), i.e. regions precisely identified by us and others to be implicated in -arrestin interaction and PKC signaling, respectively. Using this approach we also tested for the first time the function of 4 proline-rich (PxxP) motifs in the PTH1R distal C terminus that we had identified in silico.

All experiments were performed in transiently transfected HEK(Human Embryonic Kidney)293 cells. HEK293 cells were used because they do not naturally express PTH1R and are easily transfectable with cDNA of mutant receptors, -arrestin and other components of this signaling cascade. Using this cell system, our group had previously reported the role of -arrestin in PTH1R trafficking and regulation of G proteins signaling (245, 248).

To detect ERK1/2 activation, we used Western Blot for phosphorylated ERK1/2.

Western blot is a method to detect a specific protein (in our case p-ERK1/2) in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide. The proteins are then transferred to a membrane (typically nitrocellulose), where they are probed using antibodies specific to the target protein, in this case phospho-ERK1/2 at position 202 (Threonine) and 204 (Tyrosine).

In addition, to evaluate the functionality of activated ERK1/2 in this system, ERK1/2-mediated transcriptional activation was analysed by co-transfecting HEK293 cells with a SRE(Serum Responsive Element)-luciferase reporter cDNA construct encoding 5 SRE motifs. SRE is a short sequence found in the promoter regions of a number of immediate early genes including c-fos, junB, and Egr-1/2. SRE binds Serum response factor (SRF), a 508 aa protein, which can be activated by serum 66

factors, lysophosphatidic acid, TNF and other molecules. There are two main classes of signaling cascades leading to SRF-mediated activation of SRE. One is a ternary complex factors (TCF)-dependent pathway involving the ras-raf-MAPK-ERK cascade (282). Thereby, ERK1/2 have been found to activate TCFs which are Elk1, Sap-1 and Sap-2. Both phosphorylation of TCFs and the binding of TCFs to SRF are required for activation of the SRE along this pathway. On another side, a TCF-independent pathway involves the Rho family of GTPases (283).

We used co-immunoprecipitation to analyze interactions between (wild type (WT) or mutated PTH1R and (WT or mutated) molecules such as -arrestin and c-Src. Co-immunoprecipitation can identify interacting proteins or protein complexes present in cell extracts: by precipitating one protein believed to be in a complex, additional members of the complex are captured as well and can be identified. The protein complexes, once bound to the specific antibody, are removed from the bulk solution by capture with an antibody-binding protein attached to a solid support such as an agarose bead.

To eventually investigate whether ERK1/2 activation by mutated PTH receptors was related to receptor internalization and -arrestin translocation, we used GFP-tagged PTH1R and -arrestin2 and monitored traficking in response to agonists by fluorescence microscopy in living cell maintained at 37°C.

Summary of main results obtained:

1) Stimulation of PTH1R by PTH leads to:

a) ERK1/2 activation at 5 minutes

b) Increase of ERK1/2-mediated SRE-transcriptional activity

c) Recruitment of -arrestin to the receptor followed by its internalization 2) Mutating a 4 amino acids motif (EKKY<DSEL) in IC2 that prevents coupling to Gq leads to:

a) Inhibition of ERK1/2 activation by PTH

b) Absence of PTH-stimulated SRE-transcriptional activity

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c) Disappearance of -arrestin recruitment and receptor internalization 3) Mutating serine residues into alanine in the proximal receptor C terminus leads to:

a) Decrease of ERK1/2 activation at 5 min. by PTH b) Decrease of SRE-transcriptional activity by PTH

c) No recruitment of -arrestin to the receptor nor receptor internalization 4) Mutating the 4 PxxP motifs into AxxA in the distal C terminus leads to

a) Decrease activation of ERK1/2 at 5 min. by PTH b) Decreased SRE-transcriptional activity by PTH

c) Recruitement of -arrestin to the receptor but unstable -arrestin complexes in the cytoplasm

d) Disappearance of c-Src binding to the receptor, whereas co-transfection with a contitutively active c-Src restored c-Src binding and -arrestin to the receptor

Thus, our study shows that PTH activation of ERK1/2 engages at least two distinct signaling pathways which require distinct structural determinants at the PTH1R level. The first pathway, which by analogy with other GPCRs may be defined as the

‘classical’ pathway, involves PTH1R second intracellular loop (IC2) and induces the Gq/PLC/PKC/RAF-1/ERK signaling cascade. The second pathway, which we may define as the ‘terminal’ pathway, requires the PTH1R C terminus for -arrestin binding to the proximal region and c-Src binding to distal PxxP motifs in order to activate the -arrestin/c-Src/RAS/RAF-1/ERK signaling cascade. Cooperation between the ‘classical’ and ‘terminal’ pathway also occurs upstream of ERK1/2 activation, i.e. phosphorylation of serine residues by PKC for -arrestin binding and G subunit binding to the PTH1R juxta-membrane region play a role to activate c-Src.

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Proline-rich Motifs in the Parathyroid Hormone