**2.1 Receptors and ligands**

The EGF system is present in human organs and play important role in cell proliferation, differentiation and apoptosis during embryogenesis and postnatal development [Casalini et al., 2004; Uberall et al., 2008].

The EGF system has four receptors: epidermal growth factor receptor (EGFR) (also known as ErbB-1, HER1), ErbB-2 (also called HER2, Neu), ErbB-3 (also called HER3) and ErbB-4 (also called HER4)] [Holbro et al., 2003; Yarden, 2001a; Yarden & Sliwkowski, 2001b].

ErbB receptors belong to subclass I of the superfamily of Receptor Tyrosine Kinases (RTKs) [Holbro et al., 2003; Uberall et al., 2008]. They are trans-membrane glycoproteins with an extracellular region containing two ligand-binding domains, an extracellular juxtamembrane region, a hydrophobic transmembrane domain and an intracellular domain with tyrosine kinase activity [Riese et al., 2007; Yarden, 2001a; Yarden & Sliwkowski, 2001b]. They catalyze the transfer of the γ phosphate of ATP to hydroxyl groups of tyrosines in target proteins [Hunter, 1998]. ErbB-3 lacks intrinsic tyrosine kinase activity [Mass, 2004].

The extracellular region of ErbB receptors has 4 subdomains (I-IV). Subdomains I and III (also called L1 and L2) are important for ligand binding. Subdomain II (also called S1) is important for dimerization between two receptors [Ogiso et al., 2002].

The EGF system has numerous ligands. According to their affinity for one or more ErbB receptors, they divided into three groups:


The ligands for ErbB receptors bind to the extracellular domain, resulting in receptor activation by homodimer and/or heterodimer formation and the subsequent transphosphorylation of tyrosine residues in the cytoplasmic region [Alroy & Yarden, 1997; Yarden, 2001a; Yarden & Sliwkowski, 2001b ; Holbro et al., 2003]. No direct ligand for ErbB-2 has been described [Holbro et al., 2003].

#### **2.2 ErbB receptors homodimerization and heterodimerization**

The extracellular region of EGFR, ErbB-3 and ErbB-4 has two distinct conformations:


In the absence of ligand binding, the extracellular region of EGFR, ErbB-3 and ErbB-4 has equilibrium between closed and open conformation [Ferguson et al., 2003; Dawson et al., 2005; Ozcan et al., 2006; Riese et al., 2007]. This equilibrium favours the closed conformation [Ozcan et al., 2006; Riese et al., 2007].

Ligand binding stabilizes extracellular region in the open conformation and leads to the formation of both homodimeric and heterodimeric ErbB receptor complexes [Olayioye et al., 2000; Dawson et al., 2005; Ozcan et al., 2006; Riese et al., 2007]. The dimeric formation triggers receptor activation by an allosteric mechanism [Zhang et al., 2006]. That leads to intracellular kinase activation and initiation of downstream signaling pathways [Qian et al., 1994; Olayioye et al., 2000; Yarden & Sliwkowski, 2001b].

The extracellular region of ErbB-2 has a conformation not suitable for ligand binding [Garrett et al., 2003]. However, this conformation allows extension of the receptor dimerization arm in subdomain II [Burgess et al., 2003; Garrett et al., 2003; Riese et al., 2007]. This suggests that ErbB-2 is capable for ligand independent dimerization and signaling [Riese et al., 2007]. ErbB-2 heterodimerizes with other ErbB receptors and it is their preferred heterodimerization partner [Hynes & Stern, 1994; Graus-Porta et al., 1997; Olayioye et al.,

The extracellular region of ErbB receptors has 4 subdomains (I-IV). Subdomains I and III (also called L1 and L2) are important for ligand binding. Subdomain II (also called S1) is

The EGF system has numerous ligands. According to their affinity for one or more ErbB

1. The first group includes ligands with binding specificity for EGFR: EGF, transforming growth factor-a (TGF-a) and amphiregulin (AR) [Yarden, 2001a; Yarden & Sliwkowski,

2. The second group includes ligands with dual binding specificity for EGFR and ErbB4: betacellulin (BTC), heparin-binding growth factor (HB-EGF) and epiregulin (EPR) [Yarden, 2001a; Yarden & Sliwkowski, 2001b; Holbro et al., 2003; Normanno et al.,

3. The third group includes ligands with binding specificity for ErbB-3 and ErbB-4: neuregulins (NRGs) or heregulins (HRGs). They divided in two subgroups based on their ability to bind ErbB-3 and ErbB-4 (NRG-1 and NRG-2) or only ErbB-4 (NRG-3 and NRG-4) [Zhang et al., 1997; Harari et al., 1999; Yarden, 2001a; Yarden & Sliwkowski,

The ligands for ErbB receptors bind to the extracellular domain, resulting in receptor activation by homodimer and/or heterodimer formation and the subsequent transphosphorylation of tyrosine residues in the cytoplasmic region [Alroy & Yarden, 1997; Yarden, 2001a; Yarden & Sliwkowski, 2001b ; Holbro et al., 2003]. No direct ligand for ErbB-

The extracellular region of EGFR, ErbB-3 and ErbB-4 has two distinct conformations:

1. The closed conformation (inactive), has intramolecular interactions between subdomains II and IV [Ferguson et al., 2003; Dawson et al., 2005; Riese et al., 2007]. 2. The open conformation (active), where subdomains I and III form a ligand-binding pocket that permits interactions between a single ligand and subdomains I and III

In the absence of ligand binding, the extracellular region of EGFR, ErbB-3 and ErbB-4 has equilibrium between closed and open conformation [Ferguson et al., 2003; Dawson et al., 2005; Ozcan et al., 2006; Riese et al., 2007]. This equilibrium favours the closed conformation

Ligand binding stabilizes extracellular region in the open conformation and leads to the formation of both homodimeric and heterodimeric ErbB receptor complexes [Olayioye et al., 2000; Dawson et al., 2005; Ozcan et al., 2006; Riese et al., 2007]. The dimeric formation triggers receptor activation by an allosteric mechanism [Zhang et al., 2006]. That leads to intracellular kinase activation and initiation of downstream signaling pathways [Qian et al.,

The extracellular region of ErbB-2 has a conformation not suitable for ligand binding [Garrett et al., 2003]. However, this conformation allows extension of the receptor dimerization arm in subdomain II [Burgess et al., 2003; Garrett et al., 2003; Riese et al., 2007]. This suggests that ErbB-2 is capable for ligand independent dimerization and signaling [Riese et al., 2007]. ErbB-2 heterodimerizes with other ErbB receptors and it is their preferred heterodimerization partner [Hynes & Stern, 1994; Graus-Porta et al., 1997; Olayioye et al.,

important for dimerization between two receptors [Ogiso et al., 2002].

2001b; Holbro et al., 2003; Normanno et al., 2003;].

2001b; Holbro et al., 2003; Normanno et al., 2003].

**2.2 ErbB receptors homodimerization and heterodimerization** 

[Ferguson et al., 2003; Dawson et al., 2005; Riese et al., 2007].

1994; Olayioye et al., 2000; Yarden & Sliwkowski, 2001b].

receptors, they divided into three groups:

2 has been described [Holbro et al., 2003].

[Ozcan et al., 2006; Riese et al., 2007].

2003;].

2000; Yarden & Sliwkowski, 2001b; Garrett et al., 2003]. At elevated expression levels ErbB-2 homodimerizes [Garrett et al., 2003].

ErbB-3 lacks intrinsic tyrosine kinase activity and therefore can initiate signaling only in association with another ErbB receptor, usually ErbB-2 [Mass, 2004].

Although both homodimerization and heterodimerization result in activation of the EGF system network, heterodimers are more potent and mitogenic [Marmor et al., 2004]. ErbB-2 and ErbB-3 heterodimer is the most transforming and mitogenic receptor complex and increases cell motility on stimulation with a ligand [Alimandi et al., 1995; Wallasch et al., 1995; Yarden & Sliwkowski, 2001].

The dimerization of ErbB receptors represents the fundamental mechanism that drives transformation [Zhang et al., 2007].

#### **2.3 ErbB receptors signaling**

Dimerization of ErbB receptors leads to intracellular kinase activation [Olayioye et al., 2000; Qian et al., 1994; Yarden & Sliwkowski, 2001b]. As a result, a number of tyrosine residues in the COOH-terminal portion of ErbB receptors become phosphorylated [Burgess et al., 2003; Holbro et al., 2003; Zhang et al., 2007]. These phosphorylated tyrosine residues function as docking sites for cytoplasmic proteins containing Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains [Songyang et al., 1993; Marmor et al., 2004; Yarden & Sliwkowski, 2001b; Zhang et al., 2007]. Recruitment of proteins initiates intracellular signaling via several pathways:

#### **2.3.1 Ras / Raf / mitogen-activated protein kinase (MAPK) pathway**

The Ras / Raf / mitogen-activated protein kinase (MAPK) pathway regulates cell proliferation and survival [Scaltriti & Baselga, 2006]. Following ErbB phosphorylation, the complex of Grb2 and Sos adaptor proteins binds directly or indirectly (through Shc adaptor protein) to specific intracellular ErbB docking sites [Lowenstein et al., 1992; Batzer et al., 1994].

This interaction results in conformational modification of Sos, leading to recruitment of Ras-GDP and subsequent Ras activation (Ras-GTP) [Hallberg et al., 1994]. Ras-GTP activates Raf-1 and, through intermediate steps, phosphorylates MAPK-1 and MAPK-2 [Hallberg et al., 1994; Liebmann, 2001]. Activated MAPKs phosphorylate and regulate specific intranuclear transcription factors involved in cell migration and proliferation [Hill & Treisman, 1995; Scaltriti & Baselga, 2006 Gaestel, 2006].

#### **2.3.2 Phosphatidylinositol 3-kinase (PI3K) / Akt pathway**

The Phosphatidylinositol 3-kinase (PI3K) / Akt pathway regulates cell growth, apoptosis, tumor invasion, migration and resistance to chemotherapy [Vivanco & Sawyers, 2002; Shaw & Cantley, 2006].

PI3K is a dimeric enzyme that composed of a regulatory p85 subunit and a catalytic p110 subunit [Vivanco & Sawyers, 2002]. The regulatory p85 subunit, is responsible of the anchorage to ErbB receptor specific docking sites, through interaction of its Src homology domain 2 (SH2) with phosphotyrosine residues [Yu et al, 1998a; Yu et al., 1998b]. The catalytic p110 subunit, catalyze the phosphorylation of phosphatidylinositol 4, 5 diphosphate at the 3' position [Vivanco & Sawyers, 2002]. Phosphatidylinositol 3, 4, 5 triphosphate, phosphorylates and activates the protein serine/threonine kinase Akt [Stokoe et al., 1997; Vivanco & Sawyers, 2002].

ErbB receptor specific docking sites for p85 subunit are present on ErbB-3 and absent on EGFR [Carpenter et al., 1993; Yarden & Sliwkowski, 2001b]. EGFR dependent PI3K activation occurs through dimerization of EGFR with ErbB-3 or through the docking protein Gab-1 [Mattoon et al., 2004; Scaltriti & Baselga, 2006].
