Mechanism of Activation

Ligand-Induced Receptor Dimerization

Protein tyrosine kinase receptors are activated by ligand-induced dimerization in all cases that have been investigated [29]. This brings the receptor kinase domains close to each other, which results in autophosphorylation in trans within the intracellular parts of the receptors. The autophosphory-lation occurs on tyrosine residues located within or outside the kinase domain of the receptor.

There are, however, many different modes whereby ligand binding induces receptor dimerization [13]. Some ligands are disulfide-bonded dimers, such as PDGFs and VEGFs; the binding of these ligands leads to formation of a symmetric complex consisting of two receptors and one dimeric ligand [39]. In contrast, ephrins are monomeric molecules; after binding of two ephrin molecules to Eph receptors, a dimeric receptor complex is formed in which each ephrin molecule contacts two receptors and each receptor contacts two ligands [15]. Ligand binding to the EGF receptor causes a conformational change allowing direct receptor-receptor interaction which stabilizes dimerization [9a,24a]. There are also examples of accessory molecules helping to stabilize a dimeric complex; FGFs are monomeric molecules that interact with receptors at a 1:1 stoichiometry, and receptor dimerization is induced by binding of heparin or heparan sulfate to the complex of FGF and receptor [30]. Finally, members of the insulin receptor family are already disulfide-bonded dimers before ligand binding; binding of ligand presumably induces a conformational change that allows receptor autophosphorylation and activation.

Although receptor dimerization is likely to be necessary for activation of PTK receptors, it is not always sufficient. Evidence suggests that the orientation of the two intracellu-lar domains in the receptors relative to each other is important [19]. Moreover, there are indications that the initial dimerization of EGF receptors may be followed by further oligomerization, which may be necessary to obtain a fully active receptor [4].

Homo- and Heterodimerization

In the classical case of ligand-induced dimerization of PTK receptors, two identical receptors form a homodimer; however, two related receptors from the same subfamily may also form a heterodimer. Examples from the PDGF [14] and EGF [41] receptor subfamilies show that heterodimeric receptors may have quantitatively or qualitatively different signaling capacities.

There are also examples of heteromeric complexes between individual PTK receptors and unrelated receptors. Examples include interactions between PTK receptors from one subfamily with PTK receptors from another subfamily (e.g., PDGF and EGF receptor have been shown to interact [27]). In addition, PTK receptors have been shown to bind to integrins, an interaction that has been shown to enhance integrin signaling [10]. In some cases, interactions with non-kinase receptors enhance the affinity for ligand binding (e.g., a long splice form of VEGF binds to its PTK receptors with higher affinity if the receptor neuropilin is also part of the complex [34]).

Activation of the Receptor Kinase

Ligand-induced PTK receptor dimerization leads to autophosphorylation of the receptors in trans within the complex. The autophosphorylation serves two important roles: (1) it causes activation of the kinase domain and (2) it creates docking sites for downstream SH2-domain-containing signaling molecules. Autophosphorylation may lead to activation of the kinase via several different mechanisms, of which more than one may apply for individual PTK receptors [2]. Tyrosine residues exist within the activation loops of kinases; after phosphorylation, these residues cause the loop to swing out and open up the active site of the kinase [17]. Because most PTK receptors are phosphorylated in this region, this is likely to be a common mechanism for activation of the kinase domain. However, members of the EGF receptor family are not autophosphorylated in the activation loop. In these receptors, it is possible that the activation loops do not efficiently inhibit the kinase of the receptors. Instead, it has been proposed that the long C-terminal tails of these receptors block the active site of the kinase, an inhibition that may be relieved by autophosphorylation and a conformational change of the C-terminal tail, as has been shown for the PTK receptor Tek [32]. Finally, the recent elucidation of the three-dimensional structure of the Eph receptor revealed that in the inactive receptor the juxtamembrane domain forms a helical structure that distorts the small lobe of the kinase domain and prevents access to the active site of the receptor; after autophosphorylation in the juxtamembrane domain, this loop moves away and opens up the active site of the receptor [40].

Docking of SH2 Domain Signaling Proteins

The SH2 domain is a protein module that folds to form a pocket into which a phosphorylated tyrosine residue fits [25]. Genes encoding 87 SH2-domain-containing proteins with a total of 95 SH2 domains are present in the human genome [36]. They interact with phosphorylated tyrosine residues in a specific manner that is directed mainly by the three to six amino acid residues downstream of the phos-phorylated tyrosine residue.

As an example, Fig. 2 illustrates the interaction between the autophosphorylated PDGF P-receptor and different SH2-domain-containing molecules. One class of SH2 domain proteins has intrinsic enzymatic activity (e.g., the tyrosine kinase Src, phospholipase Cy, the tyrosine phos-phatase SHP-2, and the GTPase-activating protein (GAP) for Ras. The respective enzymatic activities are induced by binding of the SH2 domain to the receptor or by tyrosine phosphorylation induced by the receptor kinase; alternatively, the enzyme is constitutively active and, by binding to the receptor, may simply be brought to the inner leaflet of the cell membrane, where the next component in the signaling chain is located.

Other SH2 domain proteins are devoid of intrinsic enzymatic activity and serve as adaptors that connect the activated receptors with downstream signaling molecules. Adaptors often have additional domains that mediate interactions with other molecules, such as the SH3, PTB, and PH domains [25]. Examples of such adaptors include Nck, Crk, and Shc, as well as Grb2, which forms a complex with Sos, a nucleotide exchange molecule for Ras, and the regulatory subunit p85, which forms a complex with the catalytic subunit p110 of phosphatidylinositol 3''-kinase (PI3-kinase).

The interaction between the activated and autophospho-rylated receptor and individual SH2-domain-containing molecules initiates signaling pathways that lead to growth stimulation, survival, migration, and actin reorganization. The signaling capacity of a receptor is thus dependent on which SH2 domain proteins it can dock. Differential autophosphorylation may also be the mechanism by which

Figure 2 Schematic illustration of a complex between PDGF-BB and two PDGF p receptors. Known autophosphorylated tyrosine residues (P) and their numbers in the receptor sequence are indicated, as well as their interactions with SH2-domain-containing signaling molecules. Signaling molecules with intrinsic enzymatic or transcription factor activity are to the left, and adaptors to the right. Note that it is not known how many SH2 domain proteins can bind simultaneously to a dimeric receptor complex.

Figure 2 Schematic illustration of a complex between PDGF-BB and two PDGF p receptors. Known autophosphorylated tyrosine residues (P) and their numbers in the receptor sequence are indicated, as well as their interactions with SH2-domain-containing signaling molecules. Signaling molecules with intrinsic enzymatic or transcription factor activity are to the left, and adaptors to the right. Note that it is not known how many SH2 domain proteins can bind simultaneously to a dimeric receptor complex.

heterodimeric receptor complexes acquire unique signaling properties [14]. It should be noted that one member of the EGF receptor family, ErbB3, is devoid of kinase activity, yet in heterodimeric configuration with other members of the family it has a potent signaling capacity due to its ability to provide docking sites for SH2 domain proteins [41].

Inhibition of Phosphatases

The phosphorylation events performed by PTK receptors are counteracted by dephosphorylation by specific tyrosine phosphatases. Recent studies have shown that in order for efficient signaling via PTK receptors, tyrosine phosphatases must be inactivated [35,37]. This may be done by transient, specific oxidation of a cysteine residue in the active site of phosphatases, induced after PTK receptor activation in a PI3-kinase-dependent manner [1].

Regulated Intramembrane Proteolysis

Although activation of cytoplasmic signaling pathways by docking of SH2 domain signaling proteins is a major mode of signaling via PTK receptors, an alternative mechanism was recently revealed. The EGF receptor family member ErbB4 was shown to undergo regulated proteolysis in two steps. First, the extracellular domain is cleaved off by a metalloprotease, then another protease, y-secretase, cleaves within the transmembrane domain and liberates the intracellular domain of ErbB4 for translocation to the nucleus, where it potentially can regulate transcription directly [24]. A similar situation may prevail for the EGF receptor [23]. Although regulated intramembrane proteolysis is a well-established signaling mechanism for another receptor type (i.e., Notch), its general importance in PTK receptor signaling remains to be elucidated.

Control of PTK Receptor Activity

Receptor Internalization and Degradation

After ligand-induced receptor activation, PTK receptors are often accumulated in coated pits and thereafter internalized in endosomes [5], where they are deactivated by several different mechanisms. Upon acidification of the milieu inside the endo-somes, the ligand may dissociate from the receptor, which then monomerizes, becoming dephosphorylated by tyrosine phosphatases and then being recycled back into the membrane. Alternatively, the ligand-receptor complex is degraded after fusion of the endosomes with lysosomes. Moreover, PTK receptors have been shown to become ubiquitinated after activation. The ubiquitination may be mediated by interaction of the activated receptor with the ubiquitin ligase Cbl and may trigger degradation also in proteasomes [20,22].

Control of PTK Receptor Signaling

There are several examples of mechanisms that control PTK receptor signaling. When pathways that stimulate certain cellular responses are initiated, signals that inhibit the same responses are often induced. Examples include Ras activation by the PDGF receptor; at the same time as Ras is activated (i.e., converted to its GTP-bound form by the actions of the Grb2/Sos complex), it is also inactivated (i.e., converted to the GDP-bound form by RasGAP). The net effect on Ras activation by the PDGF receptor is thus dependent on the stoichiometry in phosphorylation of the tyrosine residues that can bind Grb2/Sos and RasGAP; evidence suggests that this balance can differ, for example, between homo- and heterodimeric receptor complexes [6].

Other examples of such mechanisms are the tyrosine phosphates SHP-1 and -2, each of which has two SH2 domains through which they can bind to several PTK receptors. The binding to tyrosine-phosphorylated residues activates the enzymatic activities of SHP-1 and -2, which may then counteract signaling by dephosphorylating the receptor or its substrates. It is an interesting possibility that SHP-1 and -2, or other tyrosine phosphatases, may dephosphorylate individual tyrosine residues with different efficiency and thereby modulate signaling not only quantitatively but also qualitatively [42].

To complicate the issue even further, evidence indicates that SHP-2 and possibly RasGAP, in addition to their negative modulatory role in signaling, also influence signaling by serving as adaptor molecules providing a bridge between the PTK receptor and downstream signaling molecules.

Another mechanism for feedback control of signaling is via activation of protein kinase C (PKC). The classical members of the PKC family are activated by Ca2+ and diacylglyc-erol, which are produced downstream of phospholipase Cy. For instance, the receptors for EGF, insulin, HGF, and stem cell factor are phosphorylated by PKC in such a way that inhibits the tyrosine kinase activities of the receptors [3].

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