Charming neighborhoods on the cell surface: Plasma membrane microdomains regulate receptor tyrosine kinase signaling

Cellular Signalling 27 (2015) 1963–1976

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Review

Charming neighborhoods on the cell surface: Plasma membrane microdomains regulate receptor tyrosine kinase signaling Ralph Christian Delos Santos, Camilo Garay, Costin N. Antonescu ⁎ Department of Chemistry and Biology, Ryerson University, 350 Victoria Street, Toronto, Ontario M5B 2K3, Canada Graduate Program in Molecular Science, Ryerson University, 350 Victoria Street, Toronto, Ontario M5B 2K3, Canada

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Article history: Received 18 May 2015 Accepted 7 July 2015 Available online 9 July 2015 Keywords: Receptor signaling Spatial organization Lipid rafts Clathrin Tetraspanin Dorsal ruffle Signal transduction

a b s t r a c t Receptor tyrosine kinases (RTK) are an important family of growth factor and hormone receptors that regulate many aspects of cellular physiology. Ligand binding by RTKs at the plasma membrane elicits activation of many signaling intermediates. The spatial and temporal regulation of RTK signaling within cells is an important determinant of receptor signaling outcome. In particular, the compartmentalization of the plasma membrane into a number of microdomains allows context-specific control of RTK signaling. Indeed various RTKs are recruited to and enriched within specific plasma membrane microdomains under various conditions, including lipidordered domains such as caveolae and lipid rafts, clathrin-coated structures, tetraspanin-enriched microdomains, and actin-dependent protrusive membrane microdomains such as dorsal ruffles and invadosomes. We examine the evidence for control of RTK signaling by each of these plasma membrane microdomains, as well as molecular mechanisms for how this spatial organization controls receptor signaling. © 2015 Elsevier Inc. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lipid-ordered microdomains and lipid rafts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Recruitment and regulation of RTKs in caveolae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Caveolae enhance IR signaling via caveolin binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Caveolins also enhance signaling of EphR, TrkA and other RTKs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. Caveolins negatively regulate EGFR signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Localization and regulation of RTKs within non-caveolar rafts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Other lipid-dependent signaling assemblies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Plasma membrane clathrin microdomains. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Many early ligand-stimulated RTK signaling events occur during receptor residence within CCPs. . . . . . . . . . . . . . . . . . . 3.2. PM clathrin structures are unique microenvironments for lipid regulation and are enriched in proteins with signal transduction capabilities. 3.3. Clathrin, but not receptor endocytosis, is required for certain aspects of RTK signaling . . . . . . . . . . . . . . . . . . . . . . . Tetraspanin-enriched microdomains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Control of RTK signaling by interactions with the tetraspanin web . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Negative regulation of RTK signaling by CD82 and other tetraspanins . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. Enhancement of RTK signaling by CD151 and other tetraspanins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Mechanisms of control of RTK signaling by the tetraspanin web and TEMs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Actin-dependent protrusive membrane microdomains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Dorsal Ruffle (DR) formation is controlled by RTKs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Dorsal ruffles regulate certain aspects of RTK signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1. RTK signaling intermediates are enriched within DRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2. Regulation of RTK signaling by DRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Regulation of RTK signaling in invadosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other microdomains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

⁎ Corresponding author at: Department of Chemistry and Biology, Ryerson University, 350 Victoria Street, Toronto, Ontario M5B 2K3, Canada. E-mail address: [email protected] (C.N. Antonescu).

http://dx.doi.org/10.1016/j.cellsig.2015.07.004 0898-6568/© 2015 Elsevier Inc. All rights reserved.

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6.1. Primary cilia . . . . . . . . 6.2. Galectin-based lattices . . . 7. Conclusions and future perspectives. Acknowledgments . . . . . . . . . . . References. . . . . . . . . . . . . . .

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1. Introduction Receptor tyrosine kinases (RTKs) are a family of transmembrane proteins that control numerous cellular functions including proliferation, differentiation, migration, adhesion, and metabolism and as such are critical regulators of development and tissue homeostasis. Furthermore, most human tumors have a disruption of at least one RTK, either as a result of mutation, genomic amplification or other alteration; these disruptions contribute to tumor growth and metastasis. With few exceptions, RTK binding to specific hormone ligands via their exofacial domains controls their intrinsic cytosolic kinase activity, in some cases as a result of enhanced receptor dimerization. Autophosphorylation of RTKs as well as the phosphorylation of secondary signaling molecules allows recruitment of cytosolic proteins that bind motifs containing phosphotyrosine residues, including phosphotyrosine binding (PTB) and Src-homology 2 (SH2) domains [1]. These proteins then engage a number of additional

Fig. 1. Receptor tyrosine kinase signaling pathways control cell physiology. Shown is a diagram depicting a summary of some of the common signal transduction pathways activated by receptor tyrosine kinases. With few exceptions, ligand binding induces receptor phosphorylation, which leads to the binding and recruitment of signaling adaptor proteins harboring phosphotyrosine binding (PTB) or Src homology 2 (SH2) domains. While some receptor-specific variations exits, RTKs can activate the following pathways: (i) Janus Kinase (JAK), which phosphorylates Signal Transducer and Activator of Transcription (STAT) family proteins, (ii) son-of-sevenless (SOS), which controls the activation of Ras and Raf GTPases, thus activating the mitogen activated protein kinase (MAPK) pathway, (iii) phospholipase C γ, which hydrolyses phosphatidylinositol-(4,5)-bisphosphate (PIP2) to produce inositol trisphosphate (IP3) and diacylglycerol (DAG), which in turn control activation of certain isoforms of protein kinase C (PKC), (iv) phosphatidylinositol-3-kinase (PI3K), which catalyzes formation of phosphatidylinositol-3,4,5-trisphosphate (PIP3), which controls the activation of Akt, thus leading to activation of other signals such as mTOR, (v) Rho-family GTPases including Cdc42, Rho, Rac and TC10, which together with other proteins such as Neuronal Wiskott–Aldrich Syndrome protein (N-WASP), cofilin and WASP-family verprolin-homologous protein (WAVE) control the dynamic remodeling of the actin cytoskeleton. Together, these signaling pathways combine and cross-regulate to effect control of cellular physiology.

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secondary signals which facilitate the activation of many signal transduction pathways, including Ras-MAPK, phosphatidylinositol-3-kinase (PI3K)-Akt-mTOR, phospholipase C (PLC), JAK-STAT, and Rho-family GTPases, thus allowing RTKs to control various aspects of cell physiology (Fig. 1) [2]. In many cases, ligand binding by RTKs also enhances receptor internalization, which can either lead to receptor degradation in the lysosome, receptor signaling from within endosomal compartment(s) or the nucleus, or eventual recycling of the receptor to the plasma membrane (reviewed by [3]). The internalization of many RTKs occurs through clathrin-mediated endocytosis, as demonstrated for the epidermal growth factor (EGF) receptor (EGFR) [4], MET [5], the Insulin (IR) and IGF-1 (IGF1R) receptors [6,7], RET [8] and several others; however, clathrin-independent endocytosis of EGFR and other RTKs also occurs under some conditions, such as through formation of dorsal ruffles or lipid-dependent pathways (reviewed by [3]). The compartmentalization of RTKs into plasma membrane versus endosomes can control the signaling output of the receptor [9–14], although this may be at least in part cell-context specific, as some studies have reported no effect on signaling by perturbation of receptor internalization to endosomes [15, 16]. These studies highlight the importance of signaling by RTKs localized within the plasma membrane. It has become clear that the plasma membrane is not a uniform membrane compartment; instead, it is highly heterogeneous, from the nano- to micro-scale and beyond, and is thus comprised of many distinct domains that for the sake of simplicity we will henceforth collectively refer to as plasma membrane microdomains (PMμDs). These membrane microdomains include those defined by lipids (e.g. lipid rafts, glycosynapses), microdomains defined by protein scaffolds (e.g. by clathrin or tetraspanins) and larger microdomains defined by interaction with the cytoskeleton. Here, we first explore the general mechanisms by which PMμDs may impact RTK signaling, and then examine the regulation of specific RTKs by various PMμDs. In general terms, there are several possible mechanisms by which recruitment of RTKs to PMμDs may control their function, as follows: (i) Clustering of RTKs within PMμDs. Certain RTKs such as EGFR exhibit trans-phosphorylation of unliganded receptors by nearby ligandbound receptors [17–19], a phenomenon which depends on EGFR density within the membrane [17]. As such, increasing the local concentration of an RTK within a PMμD may facilitate trans-phosphorylation of nearby receptors, whether the latter are ligand-bound or not. Indeed stimulation with low concentrations of EGF elicits only partial phosphorylation of the complement of cytosolic tyrosine residues in each individual ligandbound EGFR, while efficient multi-phosphorylation of each receptor occurred preferentially at higher EGF concentrations [20]. This suggests that ligand binding by an individual receptor or receptor dimer is insufficient to effect phosphorylation of a receptor's entire complement of tyrosine residues. Instead, higher order structures formed by RTKs [21], such as clustering of receptors within PMμDs, may be required to enhance the repertoire of phosphorylated tyrosine residues within RTKs upon ligand binding. Consistent with this, EphA2 receptors bind to soluble Ephrin ligands but do not elicit robust signaling unless receptors are cross-linked with antibodies [22] or signal in a juxtacrine manner [23]. These

R.C. Delos Santos et al. / Cellular Signalling 27 (2015) 1963–1976

studies show that clustering of RTKs, as would occur in given PMμD, may be required to promote specific signaling outcomes. (ii) Concentration of signaling intermediates within PMμDs. As we describe for specific microdomains below, many PMμDs have high relative local concentration of specific signal transduction proteins and lipids. This can in turn impact RTK signaling in at least two general ways: by enhancing the phosphorylation of specific tyrosine residues on the receptor or signal intermediates (e.g. by non-receptor tyrosine kinases), or by altering the protein–protein interactions of signaling proteins. While receptor tyrosine kinases largely undergo autophosphorylation on multiple tyrosine residues, non-receptor tyrosine kinases such as c-Src also enhance the repertoire of phosphorylated tyrosines on each RTK or on associated signaling proteins. Srcfamily kinases (SFKs) thus contribute to the phosphorylation of many RTKs and their downstream signaling intermediates [24]. As we will discuss below, specific SFKs are recruited to several PMμDs, suggesting local control of RTK signaling therein. Similarly, some protein–protein interactions involved in the regulation of signal transduction are of relatively low affinity and/or may be competitive with other interactions. Such interactions may only or preferentially occur upon localization of both binding partners to a particular microdomain, allowing efficient protein–protein interaction to occur on the basis of coincidence detection. Further, enrichment of specific lipids within PMμDs also underlies spatially restricted control of signal transduction. As we will discuss for each PMμD in turn, one of the general features of microdomains that are enriched in RTKs is that these often also recruit and enrich integrins. Integrins are heterodimers comprised of one of 18 α- and one of 8 β-subunits. Specific integrin dimers mediate interaction with the extracellular matrix. Integrins also regulate the activation of numerous intracellular signaling events, such as focal adhesion kinase (FAK), integrin-linked kinase (ILK), Src, PI3KAkt, and Rho family GTPases [25]. As such, integrin signaling has substantial overlap with the signals typically activated by RTKs. There are numerous examples of integrin association with RTKs, including that of α5β1 with vascular endothelial growth factor receptor (VEGFR) [26] or EGFR/ErbB3 [27], both of which enhance activation of PI3K-Akt signaling, reviewed by [25]. While the nature of the interaction of integrins with RTKs is complex and sometimes controversial, there is abundant evidence that integrins amplify RTK signaling. Hence, the enrichment of specific integrins with some PMμDs may be a common mechanism of control of RTK signaling. (iii) Impairment of negative signaling regulators within PMμDs. There are many tyrosine phosphatases that act on RTKs, including DEP-1 on EGFR [28] and VEGFR [29], PTP1B on EGFR [30], IR [31] and MET [32], and LAR on EphA2 [33]. The recruitment of RTKs to PMμDs may potentiate signaling by enhanced local production of reactive oxygen species (ROS), which in turn inhibits protein tyrosine phosphatases within a limited spatial range [34]. Furthermore, PMμDs may alter access of tyrosine phosphatases to their RTK signaling substrates by steric exclusion. For example, PTP1B is localized exclusively to the endoplasmic reticulum (ER) and dephosphorylates EGFR through ER contact sites with other membrane compartments [35]. Hence, recruitment of RTKs to PMμDs that either exclude or promote certain organellar contact sites may alter the access of a particular phosphatase to RTKs [36]. (iv) Alteration in RTK endocytosis, endosomal signaling and receptor degradation. Enrichment of RTKs within specific PMμDs is linked to receptor endocytosis, which can lead to signal termination via

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degradation in the lysosome or receptor recycling to potentiate further signaling (reviewed by [3]). Furthermore, RTK signaling is sustained within endosomes, and the regulation of this phenomenon on intracellular membranes is complex and extensively reviewed elsewhere [9–14]. While many of the PMμDs we discuss also control RTK internalization, here we focus on the mechanisms by which PMμDs directly control receptor signaling at the plasma membrane. With these general mechanisms by which PMμDs may control RTK signaling in mind, we examine several distinct microdomains: lipidordered domains, clathrin microdomains, tetraspanin-enriched microdomains, and actin-based protrusive membrane microdomains and we briefly examine other structures such as cilia and galectin lattices. 2. Lipid-ordered microdomains and lipid rafts Lipid rafts are distinct cholesterol- and sphingolipid-rich ordered lipid microdomains [37–43]. Lipid rafts are highly dynamic and variable in size ranging from 10 to 200 nm [40,44], and with lifetimes in the μs– ms to min range [45,46]. In addition to enrichment of specific lipids, these structures are also enriched in specific proteins [46–48], many of which harbor covalent lipid modifications such as long-chain saturated fatty acids, thus anchoring the protein on the inner PM leaflet. Other raft-localized proteins have glycophosphatidylinositol (GPI) anchors anchoring them on the outer leaflet of the PM, while yet others have intrinsic binding domains for sterols or other raft lipids (e.g., caveolin and flotillin) [49,50]. While lipid rafts are high heterogeneous, they can be divided into two broad groups: caveolae and non-caveolar, planar lipid rafts [51]. The former exhibits an invaginated structure marked by caveolin proteins, while the latter lacks caveolins and represents an ensemble of flatter membrane microdomains which may be further sub-classified, including some that still exhibit some curvature [46,52]. Many lipid rafts are enriched in specific signaling proteins such as RTKs, other kinases and phosphatases [41], and thus can spatially and temporally compartmentalize some aspects of RTK signaling (Fig. 2). Many experiments examining the localization of specific proteins to lipid rafts were based on monitoring solubility in non-ionic detergents at 4 °C; however, these conditions can cause aggregation of raft domains, leading to concern about use of these approaches [46,53]. We focus here largely on studies examining localization of RTKs using mostly detergent-free methods or microscopic techniques. 2.1. Recruitment and regulation of RTKs in caveolae Caveolae are invaginated, flask-shaped 50–80 nm structures that are found in many cells and are highly abundant in adipocytes, endothelial and muscle cells [54]. In addition to cholesterol and sphingolipids, caveolae are enriched in specific proteins such as caveolins (Cav1-3) that contribute to the membrane curvature of caveolae [40,55]. Cav-1 harbors a membrane-inserting hairpin loop, thus exposing the N- and C- termini to the cytoplasm [55,56]. The C-terminus of Cav-1 is palmitoylated, while the N-terminus harbors the caveolin-scaffolding domain (CSD) which facilitates interaction with other proteins [54–56]. Some proteins such as RTKs (e.g. EGFR, IR) have conserved caveolin-binding motifs [57]; however, these motifs may be located within the internal core of the protein kinase domains [51]. Nonetheless, IR associates with caveolae, as shown by biochemical cofractionation in detergent-free solutions and qualitative EM analyses [58–60], and IR phosphorylates caveolin [61,62], although other studies showed no enrichment of IR in caveolae [63]. Ultrastructural analysis revealed actin-dependent localization of IR to the neck but not the bulb of caveolae [64]. The platelet-derived growth factor receptor (PDGFR), TrkA and Ephrin receptors (EphR) similarly localized to caveolae by immunofluorescence or electron microscopy [51,57,65–68].

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R.C. Delos Santos et al. / Cellular Signalling 27 (2015) 1963–1976

Fig. 2. Control of receptor tyrosine kinase signaling within lipid-ordered membrane domains. Shown is a diagram depicting the heterogeneous lipid-ordered domains found within the plasma membrane. These domains can be grouped into flask-shaped, invaginated caveolae, that contain caveolin proteins, and planar lipid rafts that do not contain caveolins but some of which are enriched in flotillin proteins. Shown is the recruitment of RTKs to caveolae or specifically the caveolae neck structure, as well as other lipid-ordered domains. As discussed in the text, various specific receptor tyrosine kinases are selectively recruited to caveolae or planar lipid rafts. In addition, a number of RTKs can be regulated by direct binding to caveolins, either inside or outside of caveolae. Also shown is the recruitment and binding of TC10 to caveolin and the binding of Cbl-associated protein (CAP) and fibroblast growth factor receptor substrates (FRS) by binding to flotillins. Lipid-ordered domains are enriched in certain signals such as the Src-family kinases Lyn and Fyn.

2.1.1. Caveolae enhance IR signaling via caveolin binding The localization of IR to caveolae enhances receptor signaling [64,69, 70]. Insulin binding leads to IR tyrosine phosphorylation as well as that of insulin receptor substrate (IRS) adaptor proteins, leading to activation of signals such as the PI3K–Akt pathway [71]. Caveolin overexpression enhances IR kinase activity leading to IRS-1 phosphorylation [72, 73] and caveolin null mice exhibit decreased IR-dependent IRS-1 phosphorylation in skeletal muscle cells [72,74,75]. IR mutants unable to bind caveolin are not autophosphorylated and are poorly expressed on the PM [76]. Peptides corresponding to caveolin's CSD rescued IR expression levels in Cav-1 deficient MEFs and directly stimulated IR kinase activity in HEK-293T cells [72,73,75,77]. Caveolae and caveolins may also directly control Rho-family GTPases such as TC10 [78,79]. Upon insulin binding, TC10, Cbl and IR form a complex, leading to Cbl phosphorylation and recruitment to lipid raft domains [51,78,80]. Caveolin functions as a guanosine nucleotide dissociation inhibitor (GDI) for TC10, maintaining low basal activity [79]; caveolins also function as GDIs for Cdc42 [81,82]. Hence, caveolins function to control IR kinase activity as well as that of downstream signals such as Rho family GTPases. Disruption of caveolae by cholesterol chelation is widely used to probe the requirement for caveolae and lipid rafts for RTKs signaling [51]. Cholesterol depletion by methyl-β-cyclodextrin (MβCD) treatment has little effect on insulin binding, IR autophosphorylation and caveolin-1 binding in adipocytes [51,58,59], but impaired TC10 activation [83,84], and the downstream signaling to IRS and activation of PI3K-Akt and MAPK signaling pathways [51,57–59,76,85]. However, it should be noted that cholesterol chelation has broad impact on the plasma membrane, such as alteration of membrane fluidity [53].

2.1.2. Caveolins also enhance signaling of EphR, TrkA and other RTKs EphRs also exhibit signaling that is enhanced by caveolin [57,86], such that EphRs binding to caveolin-1 enhances the receptor's intrinsic kinase activity [73,87]. As was observed for IR, MCβD treatment did not impact EphR ligand binding, autophosphorylation or interaction with caveolin-1 [57]. This suggests that direct caveolin binding to the receptor, and not recruitment of the receptor to caveolae per se, may be sufficient for regulation of EphR and IR by caveolins. The nerve growth factor (NGF) receptor TrkA also interacts with caveolin in PC12 cells [67,88–91] and is recruited to caveolae upon ligand

stimulation [92], although electron tomography showed little localization of TrkA to caveolae [93]. TrkA signaling intermediates such as PI3K, Grb2, Ras, and MAPK were found in caveolae [88], and cholesterol depletion blocks NGF-stimulated TrkA autophosphorylation and MAPK activation [67,88]. In contrast, overexpression of caveolin-1 in PC12 cells diminished TrkA activation, resulting in suppressed differentiation [90]. Thus, the regulation of TrkA signaling within caveolae may be complex.

2.1.3. Caveolins negatively regulate EGFR signaling Some studies have reported EGFR interaction with caveolins and receptor localization to caveolae [94,95]. In contrast, many others have found no enrichment of EGFR within caveolae, instead suggesting that the raft-localized complement of EGFR is found in non-caveolar planar rafts [96–100]. Caveolin-1 is capable of forming immobile oligomers distinct from caveolae [101], such that caveolins may bind to and regulate EGFR signaling independently of caveolae [102]. In contrast to IR, the binding of caveolin-1 to EGFR is largely inhibitory. Mammary tumor cells lacking caveolin-1 exhibited a hyperactivation of EGFR signals such as MAPK, which was reversed upon re-expression of caveolin-1 [103]. Consistent with this, caveolin suppresses EGFR signaling, such as to MAPK [104–106] and cholesterol depletion increased ligand binding, dimerization and phosphorylation of EGFR [107]. Furthermore, caveolin-1 is required for the interaction between EGFR, the ganglioside GM3 and the tetraspanin CD82 [108]; these signals together effect PKCmediated phosphorylation on T654 and receptor internalization [108]. PDGFR is localized to caveolae, as detected by immunoelectron microscopy [66], and caveolin binding to PDGFR stabilizes an inactive receptor conformation [109]. Taken together, these studies indicate that caveolin proteins may control EGFR signaling by controlling receptor kinase activity and membrane traffic through direct interaction with caveolins outside of caveolae [101]. Caveolin binding also negatively regulates PDGFR signaling [109], although in contrast to EGFR, PDGFR was indeed detected within caveolae by immunoelectron microscopy [66]. Collectively, these studies indicate that caveolae have complex roles in regulating RTK signaling. Caveolae resident proteins such as caveolins enhance IR, EphR and TrkA activity, while suppressing EGFR and PDGFR through direct interaction, and some of these caveolin–RTK complexes may occur outside of caveolae. In addition, caveolae may propagate signaling of all RTKs by compartmentalization of signaling intermediates to potentiate certain RTK-elicited signaling pathways.

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2.2. Localization and regulation of RTKs within non-caveolar rafts Non-caveolar lipid rafts are enriched in cholesterol and sphingolipids [39–43], lack caveolins, and are highly heterogeneous, ranging in size from 10 to 200 nm [53]. Several proteins are enriched in planar lipid rafts (e.g. flotillin) [50], some of which exert structural alterations in raft morphology [77]. Hence, non-caveolar lipid rafts may be further sub-classified depending on the type of resident proteins they harbor [77,110]; however, we will discuss how non-caveolar lipid rafts control RTK signaling without explicit distinction of subtypes of these structures. While EGFR is not robustly detected within caveolae, some EGFR is found in detergent-resistant membrane fractions and exhibits proximity to GPI-anchored raft marker proteins [96–98,100,111–113]. Cholesterol depletion enhances EGF binding, dimerization and activation, enhancing EGFR phosphorylation as well as that of its effectors including MAPK [99,100,111]. However, other studies instead reported that cholesterol depletion impaired EGF-stimulated EGFR signaling to Akt and MAPK [114–116]. Hence, the nature of the control of EGFR signaling by organization into lipid rafts may be context specific, reflecting that cholesterol-dependent rafts may both enhance EGFR dimerization to promote signaling, yet also provide EGFR with a microenvironment that negatively regulates signaling [112,113,117]. RET receptor is activated by binding to glial-cell-derived neurotrophic factor (GDNF) family ligands. Upon GDNF binding, RET forms a complex with its co-receptor, the GPI-linked protein GFRα [118,119]. Removal of the GPI-anchor from GFRα altered GDNF-stimulated signaling, suggesting partitioning to raft membrane microdomains controls some aspects of RET signaling [120], such as that of the activation of raft-localized c-Src [121]. Similarly, cholesterol depletion also altered signaling by the Kit receptor [122]. The proteins and lipids enriched in non-caveolar planar lipid rafts control the formation and function of these structures. The stomatin/ prohinitin/flotillin/HflK/C (SPFH)-domain containing proteins are small, membrane associated or membrane-anchored proteins that oligomerize and localize to lipid rafts [123]. Of these, flotillins are be the best characterized and form unique microdomains for RTK signaling [50,123–125]. Upon ligand binding, EGFR interacts with and controls the phosphorylation of flotillins in HeLa cells [126,127]. Silencing of flotillin-1 impaired EGF-stimulated translocation of EGFR to lipid rafts and MAPK activation [126]. Consistent with this observation, silencing of flotillin-1 also impaired fibroblast growth factor (FGF)-stimulated MAPK and Akt phosphorylation in HeLa cells [128] as well as NGF-stimulated MAPK phosphorylation in PC12 cells [92]. FGF receptor (FGFR) and TrkA each phosphorylate the key adaptor protein FGFR substrate 2 (FRS2), thus facilitating MAPK phosphorylation. Flotillin-1 interacts with FRS2 [128– 129], and thus may contribute to enrichment of FGF- or NGF-stimulated signaling intermediates in lipid rafts. Flotillins also bind to c-Cbl associated protein (CAP) via the sorbin homology domain (SoHo) on CAP [130]. CAP forms a complex with flotillin upon NGF stimulation, and expression of a mutant CAP lacking the SoHo domain impaired NGF-stimulated MAPK phosphorylation [92], supporting a role for flotillin-enriched lipid rafts in scaffolding signal transduction by TrkA. Certain signaling intermediates activated by the IR also localize to flotillin lipid rafts. Insulin stimulation elicits the formation and membrane recruitment of a complex of c-Cbl and CAP, which by interaction with flotillin localizes to lipid rafts [78]. The Cbl/ CAP complex facilitates TC10 activation by recruitment of the guanyl exchange factor C3G. The scaffolding of these signaling intermediates within flotillin lipid rafts may thus contribute to the control of the membrane traffic of the insulin-responsive glucose transporter GLUT4 and the actin cytoskeleton in adipocytes [78,83]. The localization of RTKs to lipid rafts also provides access to many additional signaling intermediates enriched therein, including nonreceptor tyrosine kinases and actin-binding proteins [41], and many others as revealed by proteomic approaches [131]. Some of the best

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characterized of these include the Src family kinases (SFKs), all of which are myristoylated at the N-terminus. Additional palmitoylation of certain SFKs, such as Hck and Fyn (but not Src) confers localization of these specific RTKs to lipid rafts [132,133]. The localization of Lyn to lipid rafts may protect the kinase from dephosphorylation by phosphatases, thus promoting kinase activity [134]. Indeed selective impairment of the raft-localized subpopulation of SFK activity impaired cell adhesion and cell cycle progression [135], suggesting that raft-localized SFK activity has unique functions, perhaps to potentiate RTK signaling. 2.3. Other lipid-dependent signaling assemblies The signaling GTPase Ras is organized into small, ~ 9 nm plasma membrane nanodomains [136]. Ras isoforms are each farnesylated, yet differ by the presence of palmitoylation (H-Ras) or polylysine (K-Ras) domains [137]. H-Ras exhibits partial localization to cholesteroldependent lipid rafts in the GDP-bound state, but is localized preferentially within non-raft membranes in the GTP-bound form. K-Ras is found predominantly in the non-raft fraction. Interestingly, K-Ras and H-Ras are organized into largely distinct nanoclusters with unique lipid enrichment and perturbation of phosphatidylserine impairs segregation of Ras isoforms [136]. Interestingly, the Ras effector Raf was recruited largely to Ras nanoclusters, showing that organization of Ras into these domains is required for MAPK signaling [137]. Collectively, these results indicate that multiscale lipid-dependent organization of the plasma membrane, such as cholesterol-dependent lipid-ordered domains as well as phosphatidylserine-dependent Ras nanoclusters, contribute to the regulation of RTK signaling. 3. Plasma membrane clathrin microdomains Clathrin-mediated endocytosis (CME) is the principal method of internalization of receptors from the cell surface [3]. CME initiates by the recruitment of clathrin, the adaptor protein AP-2 and other cytosolic proteins to small, invaginating regions of the plasma membrane harboring various transmembrane proteins destined for internalization, termed clathrin-coated pits (CCPs). CCPs that undergo scission from the plasma membrane by the GTPase dynamin yield intracellular vesicles. Within CCPs, clathrin assembles into 50–200 nm lattice-like structures, and together with AP-2 acts as a scaffold for the recruitment of an additional ~ 30–50 cytosolic proteins. Interestingly, the majority of CCPs do not undergo scission and instead undergo disassembly at the plasma membrane [138–143]. These CCPs have been termed abortive as they do not yield successful internalization events, and reflect the existence of a checkpoint that monitors successful CCP assembly to gate endocytosis [140]. In addition to these diffraction-limited clathrin structures, some cells exhibit larger, flat clathrin assemblies at the plasma membrane, termed plaques [144] or flat clathrin lattices [145]. The existence of abortive CCPs and long-lived clathrin plaques may reflect some additional functions of clathrin assemblies at the plasma membrane separate from endocytosis. 3.1. Many early ligand-stimulated RTK signaling events occur during receptor residence within CCPs In the absence of ligand, many RTKs exhibit relatively inefficient recruitment to CCPs and a slow rate of internalization via CME [3]. For RTKs such as EGFR [4], MET [5], IR and IGF1R [6,7], and RET [8], ligand-binding induces receptor recruitment to CCPs and eventual receptor internalization via CME. The lifetime of individual CCPs is ~20– 60 s [142,146]. Further, considering that most plasma membrane CCPs are abortive and disassemble without leading to receptor endocytosis [138,139,141,142,146], individual RTKs may sequentially visit several abortive CCPs upon ligand binding and thus reside largely within CCP microenvironments for the first few minutes after ligand stimulation.

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Since many receptor-proximal signaling events such as Akt or PLCγ1 phosphorylation peak within 1–2 min of ligand addition [147], the activation of these signaling events occurs concomitantly to receptor residence within CCPs.

3.2. PM clathrin structures are unique microenvironments for lipid regulation and are enriched in proteins with signal transduction capabilities Several of the 30–50 proteins that are recruited to CCPs via interaction with clathrin or with AP-2 have functions that suggest direct regulation of signal transduction. These include non-receptor kinases such as AAK1 [148], which regulates ErbB4 signaling independently of receptor endocytosis [149] and Ack1 [150]. In addition, clathrin components interact with various key signaling adaptor proteins, including TOM1L1, which binds Src-family kinases (SFKs) [151], intersectin, which stimulates Ras activation via SOS binding, as well as CIN85, which binds PI3K and Src [152,153] (Fig. 3). In addition, plasma membrane CCPs are enriched in specific lipid kinases such as PI3K-C2α [154], which locally produces phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2) within CCPs [155], as well as 5-inositol phosphatases such as synaptojanins 1 and 2, OCRL, and SHIP2. PI(3,4)P2 and phosphatidylinositol-4-phosphate (PI(4)P) produced by these enzymes regulate various aspects of signal transduction, such as by binding Akt [156] or K-Ras [157,158]. Furthermore, phosphatidic acid (PA) regulates multiple aspects of EGFR internalization via CME [141] as well as membrane binding and activation of SOS leading to Ras activation [159]. Diacylglycerol kinase δ (which produces PA) binds to AP-2 and is thus recruited to CCPs [160]. Hence, CCPs function as lipid-enriched microdomains or structures that uniquely control the production and turnover of lipids that diffuse throughout the rest of the plasma membrane.

3.3. Clathrin, but not receptor endocytosis, is required for certain aspects of RTK signaling We have recently shown that clathrin, but not receptor endocytosis, is required for EGF-stimulated activation of Akt [161]. Perturbation of clathrin, through siRNA gene silencing, pharmacological inhibition or knocksideways silencing impaired EGF-stimulated Akt phosphorylation. In contrast, perturbation of dynamin, which allows formation of clathrin structures at the plasma membrane but not vesicle scission, was without impact on EGF-stimulated Akt phosphorylation. EGF-stimulated Gab1 phosphorylation was the most EGFR-proximal signaling event identified that exhibited dependence on clathrin, and phosphorylated Gab1 was found partly enriched within CCPs upon EGF stimulation [161]. Thus, in addition to receptor endocytosis, CCPs serve to scaffold key signaling molecules that can regulate specific signaling pathways at the cell surface. Consistent with this, flat clathrin lattices that may not readily lead to endocytic events recruit G-protein coupled receptors such as CCR5 [145] and CCPs function as signaling platforms for the activation of Gαi protein signaling by the delta opioid receptor [162]. Hence, plasma membrane clathrin structures are emerging as key signaling microdomains, and future research into the molecular mechanisms by which clathrin structures control signaling will provide valuable insight into the regulation of RTK function.

4. Tetraspanin-enriched microdomains Tetraspanins are a family of small proteins that each span the cell membrane four times and are abundant in virtually every mammalian cell [163]. Tetraspanins readily exhibit homo- or hetero-multimerization with other tetraspanins, forming an interaction network termed the

Fig. 3. Clathrin-coated pits are unique microdomains for the regulation of receptor tyrosine kinase signaling. Many specific RTKs undergo internalization via clathrin-mediated endocytosis upon binding to ligand. Shown is the recruitment of ligand-bound RTKs to clathrin-coated pits, invaginated membrane structures formed by binding of the heterotetrameric adaptor protein AP-2 and the assembly of clathrin into a lattice-like structure on the inner leaflet of the plasma membrane. Some clathrin-coated pits eventually undergo scission from the plasma membrane to form intracellular vesicles. Clathrin and AP-2 collectively bind and recruit between 30–50 cytosolic proteins. Shown are proteins that bind AP-2 or clathrin (directly or indirectly) that have roles that suggest function as regulators of signaling, including TOM1L1, Src-family kinases (SFKs), class II phosphatidylinositol-3-kinase (PI3K-C2α), activated Cdc42 kinase 1 (Ack1), intersectin 1 (ITSN), diacylglycerol kinase δ (DGKδ), synaptojanin 1 (Sjn1), Cbl-interacting protein of 85 kDa (CIN85). Collectively, these signals contribute to the activation of specific signals, such as the activation of PI3K and Akt upon EGF stimulation.

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tetraspanin web. This tetraspanin web underlies the formation of tetraspanin-enriched microdomains (TEMs) within the plasma membrane [163]. Tetraspanins are able to simultaneously bind other tetraspanins and receptors such as RTKs and integrins, as well as interact with and locally enrich certain lipids and recruit cytoplasmic signaling proteins. These abilities of tetraspanins underlie the role of plasma membrane TEMs in the regulation of RTK signaling. There are more than 30 human tetraspanins that contribute to formation of the tetraspanin web (reviewed by [163]). The most studied tetraspanins are those that have some antigenic properties, including tetraspanin 24 (CD151), 27 (CD82), 28 (CD81), 29 (CD9) and 30 (CD63). The molecular and structural basis of tetraspanin–tetraspanin interactions are poorly understood, although these likely involve interactions of cytoplasmic or extracellular regions of these proteins, as has been reported for CD81 [164]. Immunoprecipitation of tetraspanins in nonionic detergents such as Brij-96 (preserving the tetraspanin web) has allowed identification of many other proteins that associate with tetraspanins [165–167]. These include cell adhesion proteins such as many specific integrins, and other proteins such as CD9P-1 and EWI-2 [163], which also bridges interaction with the actin cytoskeleton [168]. The organization of the tetraspanin web into discrete TEMs within the plasma membrane is evinced by a number of studies using a variety of microscopy approaches. Tetraspanins form punctate structures at the cell surface as observed by fluorescence microscopy [169]. Immunoelectron microscopy detecting CD63 in HeLa cells revealed the existence of tetraspanin clusters with a diameter of ~200 nm2, although these were highly heterogeneous [170]. Importantly, TEMs are largely functionally and spatially distinct from lipid rafts (reviewed by [171]), demonstrated by: 1) their distinct sensitivity to cholesterol chelation and treatment with nonionic detergents such as Brij-96 [172], 2) their virtually complete lack of shared associated proteins detected by proteomic approaches [131,165,166], and 3) the lack of colocalization of tetraspanins with lipid rafts markers such as GPI-anchored proteins [169,173]. Despite being largely distinct from lipid rafts, TEMs associate with specific lipids, in particular glycosphingolipids (GSLs), such as GM2 and GM3 [174]. While TEMs have lifetimes on the order of minutes or longer, the partitioning of tetraspanin web components within TEMs is very dynamic. About one-third of CD9 and CD151 exhibited unconfined diffusion, while the tetraspanins that exhibited confined motion consistent with TEMs exhibited dwell times of mostly between 3 and 20 s [169, 173]. In addition to plasma membrane localization, many tetraspanins undergo internalization and are found on a number of intracellular organelles, including multivesicular bodies and lysosomes (reviewed by

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[175,176]). Here, we focus on the role of tetraspanins and TEMs at the plasma membrane in the regulation of RTK signaling (Fig. 4).

4.1. Control of RTK signaling by interactions with the tetraspanin web Numerous studies have demonstrated that various RTKs can be immunoprecipitated with tetraspanins, including: c-kit with CD9, CD63 or CD81 [177], Met with CD82 [178] and with CD151 [179], and EGFR with CD9 [180] and with CD82 [181,182]. Other studies demonstrated interaction of tetraspanins and RTKs in situ, such as the interaction of VEGFR and CD63 detected by proximity ligation [183]. Importantly, RTK association can be regulated by binding to ligand, such as dissociation of CD82 and EGFR upon stimulation with EGF [181]. RTKs are thus conditional constituents of the tetraspanin web. The regulation of RTK interaction with tetraspanins can elicit either positive or negative regulation of RTK signaling.

4.1.1. Negative regulation of RTK signaling by CD82 and other tetraspanins Numerous studies found negative regulation of RTK signaling by specific tetraspanins. CD82 overexpression suppresses Met signaling leading to activation of Rho family GTPases, thus impacting cytoskeletal dynamics and cell migration [178,184], and activation of PI3K and Ras [178]. The fraction of c-Kit that co-precipitated with CD9, CD63 or CD81 had impaired ligand-stimulated receptor phosphorylation [177]. Consistent with this, EGFR signaling was impaired by CD82 overexpression [181] or by antibody-induced clustering of CD9 [180].

4.1.2. Enhancement of RTK signaling by CD151 and other tetraspanins In contrast to the suppression of RTK signaling by CD82 and other tetraspanins, CD151 in particular largely enhances RTK signaling. Silencing of CD151 impairs Met signaling leading to association with Gab1 and activation of the MAPK pathway [185], as well as Met-driven proliferation [185] and migration [186]. The alteration of Met signaling by CD151 silencing occurred as a result of disruption of CD151-dependent Met association with α3/α6 integrins [186] or β4 integrin [185]. Similarly, VEGFR associates with β1-integrin and CD63, and CD63 attenuates VEGFR signaling leading to phosphorylation of FAK, Src, Akt and MAPK [183]. Also, CD151 controls ErbB2 signaling and the ability of the receptor to drive tumor growth and metastasis, possibly via control of receptor interaction with α6β4 integrins [187]. Hence, in contrast to CD82, some tetraspanins such as CD151 enhance RTK signaling.

Fig. 4. Tetraspanin-enriched microdomains recruit some receptor tyrosine kinases and regulate signaling. Shown are tetraspanins and the formation of tetraspanin-enriched microdomains (TEMs), which are formed by tetraspanin–tetraspanin interactions. TEMs are dynamic, with robust exchange of tetraspanins between highly mobile diffusive population and more static populations that likely represent TEMs. Tetraspanins interactions form the basis of a tetraspanin web, which leads to recruitment of many specific proteins and lipids, including glycosphingolipids, phosphatidylinositol-4-kinase (PI4K), protein kinase C (PKC), CUB domain-containing protein-1 (CDCP-1), and Src-family kinases (SFKs). Also shown is the facilitation of juxtacrine signaling by components of TEMs, including glycosphingolipids. Some proteins enriched within TEMs, such as integrins, are not shown in this image, but are discussed in the text.

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4.2. Mechanisms of control of RTK signaling by the tetraspanin web and TEMs There are several possible mechanisms by which RTK entanglement within a tetraspanin web and/or recruitment to TEMs can modulate RTK signaling, as follows: (i) TEMs as platforms for recruitment of cytosolic signaling intermediates. In addition to interaction with RTKs, many tetraspanins also associate with cytosolic proteins and thus may locally enrich specific signaling molecules within TEMs. Several tetraspanins, including CD9, CD81, CD82 and CD151 associate with protein kinase C (PKC) [188], and the local enrichment of PKC controls RTK signaling. Indeed PKC phosphorylates EGFR at T654 [189], a phenomenon enhanced by CD82 and GM3 [108], thus controlling EGFR tyrosine phosphorylation [108] and Cbl-dependent ubiquitinylation [190]. Tetraspanins may also represent unique microdomains for localized remodeling of phosphoinositides that impact RTK signaling. Many tetraspanins, including CD9, CD63, CD81, CD82 and CD151 bind to phosphatidylinositol-4-kinase (PI4K) [191–194], a lipid kinase that catalyzes the synthesis of PI4P [157]. PI4P recruits proteins harboring a polybasic motif to the plasma membrane, such as the signal transduction protein K-Ras [158], and Shc [193], and the activation of the MAPK pathway by CD81 cross-linking was ablated by expression of a Shc mutant deficient in PI4P binding [193]. PI4P is also used as a substrate to synthesize other important signaling lipids such as PIP2 and phosphatidylinositol-3,4,5-trisphosphate (PIP3). Indeed CD82 overexpression reduced PIP2 levels, which correlated with a disruption of Rho GTPase signaling and actin cytoskeleton dynamics [195]. From these studies emerges a model of localized control of PI4P synthesis within TEMs that controls various signaling pathways activated by RTKs, although PI4P is also synthesized elsewhere, such as in the Golgi [157]. Additionally, CD9 associates with CUB domain-containing protein-1 (CDCP1) [166,196], an adaptor that binds and regulates several kinases including c-Src and PKCδ [197,198]. Indeed CDCP1 contributes to EGF-stimulated cell migration [199]. Importantly, the association of CDCP1 with tetraspanins modulates the ability of CDCP1 to control Src activity, evinced by the observation that CD82 overexpression blocks CDCP1-enhanced Src activation [200]. Hence, TEMs represent a unique RTK signaling microenvironment as a result of enrichment of specific proteins and local synthesis of certain signaling lipids. (ii) TEMs regulate RTK association with other microdomains and alter RTK endocytosis. Tetraspanins exhibit specific and complex internalization, such as the clathrin-dependent internalization of CD63 [201] or clathrin-independent internalization of CD63 [202], CD81 [176], CD82 [203] and CD151 [204], reviewed by [176]. As many tetraspanins undergo specific recruitment to these various endocytic structures, the association of RTKs with tetraspanins or with TEMs may alter the parameters of RTK internalization, thus altering RTK signaling specificity, duration and magnitude. Overexpression of CD82 results in increased internalization of EGFR [181], as does antibody-induced clustering of CD9 [180]; each of these correlated with alterations in EGFR signaling. CD82 overexpression impairs EGFR dimerization required for signaling and alters the distribution of EGFR within the plane of the plasma membrane, favoring EGFR enrichment towards the cell margins [182]. Consistent with this, silencing of CD82 altered EGFR diffusion, increased EGFR localization to clathrin structures, and impaired early EGF-stimulated MAPK phosphorylation [205]. Furthermore, CD82 negatively regulates ligand-stimulated EGFR ubiquitinylation and c-Cbl tyrosine

phosphorylation [190]. Hence, tetraspanins such as CD82 control EGFR distribution within the PM and impair recruitment of EGFR to other PMμDs, thus altering EGFR signaling and internalization. (iii) Tetraspanins, glycosphingolipids (GSLs) and control of RTK signaling. In addition to association with specific proteins, tetraspanins also interact with specific lipids, in particular GSLs. Indeed virtually all GSLs found within a cell are within PMμDs, many of which appear to overlap with TEMs [206–208]. Together with the recruitment of proteins such as integrins, the ability of specific GSLs found in TEMs to mediate interactions such as with the surface-exposed proteins of other cells to form cell–cell contacts underlies the function of at least a subset of TEMs as microdomains termed glycosynapses [209,210]. Hence, GSL-enriched TEMs control RTK signaling as a result of lipidic interactions within a single microdomain in a particular cell, or may provide RTKs localized therein with a unique profile of juxtacrine ligands on other cells. Tetraspanins interact with GSLs, including the direct interaction of CD82 with GM2 [211]. The interaction of GSLs with tetraspanins alters the interactions of tetraspanins with other proteins, such as of CD9 with α3 integrin [212], indicating that GSL incorporation into TEMs controls their protein complement. Indeed GSLs also contribute to the enrichment of cytoplasmic proteins such as c-Src to PMμDs [206]. In addition to control of the complement of signaling proteins within TEMs, GSLs can also directly control RTK function. GM3 directly inhibits ligand-dependent EGFR kinase activation [213,214]. Consistent with this, CD82 overexpression impaired EGFR and MET signaling, and this was dependent on GM2/GM3 [201,215]. GM3 also contributes to CD82-dependent enhancement of EGFR endocytosis by recruitment of PKC [108]. Furthermore, GM2 association with CD82 impaired HGF-stimulated MET signaling [211]. As such, the interactions of RTKs with GSLs or the GSL-dependent regulation of the tetraspanin web allows the enrichment of GSLs within TEMs to play a key role in the regulation of signal transduction. The ability of TEMs and glycosynapses to mediate cell–cell contacts also suggests that these microdomains may play a role in controlling juxtacrine signaling of certain RTKs. EGFR has several ligands such as HB-EGF and TGFα, which can signal either as soluble ligands following proteolytic cleavage of a transmembrane precursor by metalloproteases or in a juxtacrine manner requiring cell–cell contact [216]. Both juxtacrine signaling as well as cleavage of soluble hormone products activate EGFR in a highly spatially restricted manner [217]. Hence, regulation of RTK localization within TEMs at sites of cell–cell contacts and regulation of proteolytic cleavage of transmembrane ligands within TEMs contribute to regulation of RTK signaling. Interestingly, CD9 impaired the proteolysis of TGFα, and the CD9-dependent increase in transmembrane TGFα increased EGFR phosphorylation and signaling in juxtacrine assays [218]. Hence, TEMs may also play an important role in the control of RTK signaling by controlling cleavage of soluble RTK ligands or by controlling juxtacrine signaling. 5. Actin-dependent protrusive membrane microdomains A number of distinct types of membrane microdomains are formed by the dynamic remodeling of the actin cytoskeleton in close apposition to the plasma membrane, including dorsal ruffles, peripheral ruffles, lamellipodia, and invadosomes. These structures are formed or regulated upon RTK stimulation, and a some control RTK signaling by recruitment and spatial organization of RTKs themselves or of a subset of their signaling intermediates. Despite some similarities, these actin-dependent protrusive structures differ in morphology, lifetime, and regulation [219, 220]. Here, we focus on the actin-dependent microdomains that are

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Fig. 5. Specific RTKs control formation of dorsal ruffles (DRs) that in turn control receptor signaling and internalization. Ligand binding by specific RTKs coordinates dramatic actin rearrangements that lead to formation of a number of protrusive actin-based structures such as dorsal ruffles in a cell- and condition-specific manner. Shown are the signals activated by RTKs that control dorsal ruffle formation, including cortactin (cort), Rac, Neuronal Wiskott–Aldrich Syndrome protein (N-WASP), WASP-family verprolin-homologous protein (WAVE), dynamin (dyn), p21-activated kinase (PAK1). A number of signaling proteins are recruited to dorsal ruffles, some of which further regulate dorsal ruffle formation: protein kinase C λ (PKCλ), phosphatidylinositol-3-kinase (PI3K, which controls the formation of phosphatidylinositol-3,4,5-trisphosphate, PIP3), Tandem PH Domain-Containing Protein 1 (TAPP1), SH3 And SYLF Domain Containing 1 (SH3YL1), Cbl, Cyld, Src, Grb2-associated binder (Gab1), protein tyrosine phosphatase 2A (PP2A), protein tyrosine phosphatase 2C (PTP2C), Abl, NADPH oxidase (NOX). PP2A and PTP2C contribute to dorsal ruffle collapse, which leads to internalization of RTKs to endosomes containing early endosome antigen 1 (EEA1).

enriched in specific RTKs and control RTK-proximal signaling, namely dorsal ruffles and invadosomes (Fig. 5).

5.1. Dorsal Ruffle (DR) formation is controlled by RTKs DRs are actin-dependent membrane-associated domains ranging in size from 5 to 15 μm that form in response to RTK ligand binding [221]. DRs are heterogeneous and can be circular, linear and/or wavelike and are observed in numerous cell types and occur upon ligand binding by RTKs, such as EGFR, PDGFR, VEGFR, MET and IR [222,223]. Formation of DRs is time-dependent, appearing b 5 min after stimulation with RTK ligands and persisting for an average lifetime of 10– 20 min [224,225]. DR collapse is linked to internalization, as evinced by the formation of a tubulo-vesicular network on the dorsal surface of Panc1 cells [224]. DR are devoid of protein markers for clathrinmediated endocytosis or caveolae [224], and some studies have instead indicated that DR collapse is clathrin-independent [224] and linked to RTK-stimulated micropinocytosis [226,227]. DR formation is dependent on dynamic actin polymerization and remodeling, mediated by the recruitment of the Arp2/3 complex therein [219,228]; DRs also require or are enriched in other actin cytoskeleton regulators such as dynamin, cortactin, N-WASP, WAVE1, Rac, Rab5, actinin-4, RN-tre, ARAP1, Arf1 and 5 and Arl13b (Fig. 5) [219,221,222, 228–230] . RTK-induced DR formation does not occur in all cell types. Indeed, exogenous expression of certain proteins such as Gab1 or Pak1 in HeLa cells enhances formation of DRs upon HGF or PDGF stimulation [231], and substrate stiffness enhances the formation of DRs in response to PDGF in vascular smooth muscle cells [232]. DRs have been proposed to contribute to the rapid remodeling of cells with robust stress fibers to allow a transition to a migration phenotype [222,230]. This suggests that the cell-type specificity for DR formation may reflect expression of specific cellular proteins or cell growth conditions. Moreover, while some RTKs can localize to DRs upon ligand binding [224,225], this recruitment is receptor-specific, as EGF stimulation elicited the recruitment of EGFR but not transferrin receptor or PDGFR to DRs [224]. DRs contribute to the internalization of specific RTKs. Fluorescentlytagged EGF can be observed within puncta emanating from dorsal ruffle structures, consistent with internalization of EGFR at or near DRs [224]. Similarly, MET is recruited to DRs in MDCK cells, and MET is co-localized with the endosomal protein EEA1 at the base of collapsing ruffles [225], suggesting MET endocytosis at these sites. Indeed, enhancement of MET-dependent formation of DRs by overexpression of Gab1 or Pak1 increased MET internalization and degradation [225].

Hence, RTK recruitment to DRs results in their sequestration from other PM regions, and eventual RTK internalization upon DR collapse [222]. As such, specific RTKs may reside within DRs for some or all of the lifetime of DRs (10–20 min), thus allowing localized control of RTK signaling within this specialized PMμD during this time.

5.2. Dorsal ruffles regulate certain aspects of RTK signaling 5.2.1. RTK signaling intermediates are enriched within DRs RTKs localized within DRs are phosphorylated and have active kinase domains [224,225], suggesting RTKs signal specifically within DRs. Indeed many proteins with signal transduction properties are enriched within DRs [219,222]. These include cytoskeletal remodeling proteins required for the formation of DRs (as discussed above), and also specific lipids with roles in signal transduction, several serine/threonine and tyrosine kinases, signaling adaptors, protein phosphatases, and their effector(s). It should be noted that DRs are likely cell-type specific and heterogeneous; thus, the lipids and proteins recruited to DRs discussed below may not all be found within all DRs concurrently. Several phosphoinositides are enriched in DRs, each of which has role(s) in signal transduction. Class I PI3K (p85 and P110α subunits) is recruited to DRs formed up insulin stimulation in L6 myotubes [223,233]. This allows local production and enrichment of PIP3 and Akt activation in membranes within these structures [223,233]. DRs formed upon EGFstimulation of A431 cells exhibited enrichment of PIP2 and PIP3 [234], as did ruffles formed in macrophages in response to M-CSF [235]. The adaptor protein SH3YL1, which binds PIP3, was recruited to dorsal ruffles upon PDGF stimulation in NIH3T3 cells [236]. SH3YL1 recruits the lipid phosphatase SHIP2, resulting in enrichment of PI(3,4)P2 within DRs. In addition to activation of specific signals within DRs (e.g. Akt), PIP3 is important for formation of DRs, as PI3K inhibitors block DR formation [237] and the phosphoinositide phosphatase PTEN negatively regulates PDGFinduced circular dorsal ruffle formation in rat aortic smooth muscle cells [238]. A related signaling module comprised of Grb2 and Gab1 is also recruited to DRs. Upon stimulation of MDCK or mouse fibroblast cells, the recruitment of Nck to Gab1 allows activation of Rac1, effecting N-WASPdependent actin remodeling for DR formation [225,237]. Gab1 is a multivalent signaling adaptor capable of recruitment of PLCγ1, PI3K and SHP-2 [239]; hence, Gab1 enrichment within DRs may be related to that of PI3K. There are several non-receptor protein kinases that can be recruited to DRs. Consistent with the activation of Rac1 within DRs [225,237], Racactivated p21-activated kinase 1 (PAK1) is recruited to dorsal ruffles in Swiss 3T3 cells upon stimulation with PDGF [231]. The atypical PKCλ is

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recruited to insulin-stimulated DRs in L6 myotubes [233] and PKCλ is required for formation of DRs upon stimulation of MEFs with PDGF [240]. PDGF stimulation of MEFs resulted in recruitment of several Srcfamily kinases to dorsal and peripheral membrane ruffles [241], and cSrc was recruited to DRs upon stimulation of NIH 3T3 cells with PDGF [242]. Src phosphorylates the deubiquitylating enzyme Cyld, and Cyld is recruited to DRs upon stimulation of fibroblasts with EGF [243]. Interestingly, Src also phosphorylates the E3-ubiquitin ligase Cbl, and Cbl is robustly localized to DRs upon PDGF stimulation of NIH-3T3 cells [244]. Hence, DRs may be sites of dynamic Src-dependent cycles of ubiquitinylation and de-ubiquitinylation of various component proteins. Another tyrosine kinase, Abl, is required for formation of DRs in primary fibroblasts [245] and in NIH-3T3 cells [242], suggesting it may also be localized within DRs. Furthermore, Abl controls recruitment of the NADPH oxidase subunit p47Phox to DRs upon PDGF stimulation in NIH3T3 cells [246] and p47Phox was also recruited to VEGFRstimulated DRs [247]. Several phosphatases can also be enriched within DRs, including PP2A and PTP2C [248,249], and PTP2C contributes to termination of DRs [249]. PP2A regulates heat shock protein-27 (Hsp27) phosphorylation within DRs in smooth muscles cells (SMC), which in turn controls induction of apoptosis and NFκB signaling [248]. Hence, DRs are microdomains enriched in specific lipids and proteins that undergo coordinated remodeling over their 10–20 min lifetimes. 5.2.2. Regulation of RTK signaling by DRs The recruitment of numerous specific signaling intermediates to DRs suggests that DRs may be functionally required for and/or regulate signal transduction. DRs may thus function to limit diffusion of proteins, thus forming a positive feedback loop for activation of specific signaling intermediates such as Rac1 [250]. Indeed the actin-disrupting agent Latrunculin B prevented the activation of Rac1 following M-CSF stimulation [250]. MET localizes to DRs upon HGF stimulation in MDCK cells and inhibition of DR formation blocks HGF-induced kidney morphogenesis [237]. In human umbilical vein endothelial cells (HUVECs), VEGF stimulation elicits DR formation and JNK phosphorylation, which required NADPH oxidase, WAVE and PAK-1 [247]. Expression of a kinase-dead Pak1 mutant or incubation with an antioxidant inhibitor of NADPH oxidase inhibited VEGF-induced DR formation and JNK2 activation [247], suggesting that DRs are required for VEGF-stimulated JNK2 activation in HUVECs. Signaling from DR microdomains is required for PI3K- and Aktdependent insulin signaling in muscle cells, in turn regulating the membrane traffic of the facilitative glucose transporter GLUT4. Inhibition of DR formation with actin disrupting small molecules or perturbation of Rac prevented the efficient recruitment of PI3K [223,251]. The remodeled actin cytoskeleton within DRs in myotubes may thus function as a “mesh” to restrict the distribution of PIP3, thus allowing activation of Akt [233]. Interestingly, while some protein components of DRs can also be recruited to other PMμDs, the pool(s) recruited to DR may have specific function(s). For example, PDGF stimulation results in recruitment of cSrc to both caveolae and to DRs, and PDGF-induced formation of DRs requires c-Src [242,252]. Importantly, DR formation is directed by a distinct pool of c-Src that is insensitive to cholesterol depletion, thus not regulated by caveolae. Instead, activation of the DR-specific pool of Src upon PDGF stimulation requires PLCy1 and sphingosine-1-phosphate (S1P)-dependent signaling pathways which are not required for mitogenic PDGF signaling [242]. Therefore, this suggests the existence of separate Src-directed signaling cascades within distinct PMμDs, each with distinct functions. 5.3. Regulation of RTK signaling in invadosomes Invadosomes is a term used to describe both podosomes (in vascular or myelomonocytic cells) and invadopodia (in cancer cells), which have

specific functions in the degradation of extracellular matrix and thus cell migration [253]. Like DRs, invadosomes are also actin-dependent structures that range from 5 to 25 μm in size and require many of the same factors that remodel the actin cytoskeleton as dorsal ruffles (e.g. NWASP, Arp2/3, cortactin, dynamin, gelsolin and cofilin, reviewed by [253]). However, invadosomes are distinct from DRs due to their positioning vis-à-vis the extracellular matrix and their high level of enrichment of matrix metalloproteases (MMPs) [219,254,255]. Despite these differences, invadosomes, specifically invadopodia, can be also enhanced by stimulation of specific RTKs. For instance, EGF stimulation induces invadopodia formation in MDA-MB-231 cells, which requires Arg-dependent cortactin phosphorylation [256]. HGF stimulation also elicits invadopodia formation following cortactin recruitment via binding to Gab1 [257]. While many studies have examined the specific recruitment of various RTKs to DRs, relatively few studies have similarly examined the recruitment of RTKs to invadosomes. Recent work has begun to elucidate that specific RTKs are indeed recruited to invadopodia. Upon HGF stimulation of MKN45 human gastric cancer cells, MET is recruited to cortactin-positive invadopodia [257]. EGFR was also found enriched within invadopodia in MDA-MB-231 and HT1080 cells [258]. Interestingly, the recruitment of EGFR and Src to invadopodia required SNARE-dependent vesicle traffic, suggesting that delivery of EGFR to invadopodia requires internalization and recycling, rather than lateral sequestration of the receptor within the plasma membrane [258]. Consistent with a requirement for EGFR localization to nascent structures for invadopodia formation, perturbation of specific SNAREs impaired invadopodia formation [258].

6. Other microdomains In addition to the PMμDs that we have examined thus far, several additional microdomains control receptor signaling, although these microdomains may not be found in all cell types.

6.1. Primary cilia Cilia are polarized microtubule-based protrusions found in quiescent cells that extend from the cell body into the extracellular space [259,260]. In contrast to motile cilia that function primarily in moving mucus and fluids, quiescent cells have a single non-motile cilia or primary cilia (PC) [259]. PCs are sensory organelles and signaling domains [260] that are comprised of a nucleated region at the basal body just beneath the plasma membrane (the cilia pocket), and the core axoneme, a structure enveloped by a lipid bilayer that is continuous with the plasma membrane [260,261]. The PC membrane is enriched in lipids and receptors providing a distinct highly regulated microdomain for signaling events which are relayed from the extracellular space into the cell [260,262]. Several RTKs are enriched within PCs, including PDGFR, IGF1R, and EGFR [262–264]. While PDGFRαα homodimers localize to the PC, PDGFRββ homodimers localize to the cell body, demonstrating that RTK recruitment to the PC is highly specific [264]. Expression of various mutants that impair PC function and transport resulted in an impairment of Akt phosphorylation by PDGFRαα [265]. Similar strategies resulting in disruption of PCs impaired IGF1R-dependent Akt phosphorylation in 3T3-L1 preadipocytes [262]. Within PCs, EGFR forms a complex with OFD1 (oral–facial–digital syndrome type 1) and flotillin proteins [263], suggesting that PCs are also enriched in this lipid raft subtype of signaling microdomain. While we provide an overview of some recent studies that demonstrated unique regulation of RTK signaling within PCs, we direct the reader to some recent comprehensive reviews on signaling within PCs [260,261].

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6.2. Galectin-based lattices Many RTKs such as EGFR have N-linked glycans, which allows interaction with lectins. One such lectin, Galectin-3, can oligomerize, leading to the formation of a galetcin lattice preferentially enriched in RTKs with specific branched N-glycans, such as EGFR [266,267]. Perturbation of Nacetylglucosaminyltransferase (GnT-V, also known as Mgat-5), which prevents the formation of N-glycans preferred by Galectin-3, impaired EGF-stimulated signaling, such as that leading to SHP-2 phosphorylation [268]. Moreover, perturbation of GnT-V dramatically enhances the rate of EGFR endocytosis [266] and also increases EGFR diffusion and interaction with Cav1, thus limiting EGFR signaling [269]. This indicates that galectin lattices restrict EGFR diffusion at the cell surface and limit its incorporation into other PMμDs. Recently, it was uncovered that Galectin-3 binding by EGFR and incorporation into galectin lattices was required for EGF-stimulated activation of RhoA, thus leading to DR formation and control of cell migration [267]. The galectin lattice also incorporates specific integrins, depending on their N-glycans, indicating that these structures may also facilitate RTK signaling by promoting signal amplification with that of integrins [267]. In addition to the domains that we have discussed here, there are many other membrane microdomains that may contribute to control of receptor signaling, including cell–cell junctions and microvilli. Furthermore, the emerging insight into plasma membrane contacts sites with other organelles indicates that these may also represent unique receptor signaling microdomains. For example, IGF1R was found within plasma membrane invaginations which contact the nucleoplasmic envelope, thus controlling Ca2+ signaling selectively in the nucleus [270]. While we have limited our discussion here to lipid-ordered PMμDs such as lipid rafts, clathrin structures, tetraspanin-enriched microdomains, dorsal ruffles and invadosomes, cilia and galectin-based structures, many of the mechanisms of regulation that we discuss likely have parallels in regulation of RTK signaling in other microdomains. Moreover, while we focus here on RTKs, many parallels can also be drawn about the role of PMμDs in the regulation of other signaling complexes, such as immune receptors and G-protein coupled receptors.

7. Conclusions and future perspectives There is abundant evidence that many RTKs localize to a number of different PMμDs, and that receptor localization within these structures imparts upon them unique signaling properties. However, this recruitment to specific microdomains appears to be controversial for some types of RTKs, such as EGFR, in that ligand binding induces recruitment to lipid-ordered domains, clathrin-coated pits, TEMs and dorsal ruffles. How might one explain this apparent contradiction of one specific type of RTK being recruited to several different PMμDs? (i) Unique properties of specific cell types. An obvious possibility is that specific cell types have intrinsic properties that allow selective routing of a particular RTK to a given microdomain. This could occur as a result of abundance of a given PMμD in a cell type, or by cell-specific receptor routing mechanisms. Consistent with this interpretation, actin-dependent protrusive microdomains such as dorsal ruffles are restricted to certain cell types and potentiated by Gab1 expression [231] and are impacted by substrate stiffness [232]. Moreover, skeletal and heart muscle express high levels of Cav-3 [39,40,271], suggesting unique abundance or function of caveolae in these cells. These findings indicate that cell type differences indeed may contribute in part to the ability of certain RTKs to partition into specific PMμDs upon ligand binding. (ii) RTKs exist in equilibrium between different microdomains. For an RTK such as EGFR that can partition into several distinct PMμDs upon ligand binding, the distribution of each receptor

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type within various microdomains controls signaling outcome. Hence, different subpopulations of EGFR may be recruited to specific PMμDs, such that microdomains “compete” for RTKs recruitment. Indeed silencing of the tetraspanin CD82 resulted in increased association of EGFR within plasma membrane clathrin structures [205]. In a series of seminal studies by di Fiore & col., the distribution of ligand-bound EGFR within plasma membrane clathrin structures and cholesterol, raft-like structures was shown to depend on the concentration of EGF ligand [272,273]. At low [EGF], the receptor localized primarily to clathrin structures, while at higher [EGF], EGFR is efficiently ubiquitinylated and routed to raft membranes devoid of clathrin [20]. Hence, while a particular receptor such as EGFR may be localized to lipid rafts, clathrin structures or TEM, there are specific conditions that facilitate preferential recruitment to a particular microdomain, thus allowing context-specific signaling. (iii) Certain PMμDs may exhibit spatial and compositional overlap. While there is abundant evidence that lipid rafts, clathrin structures, TEMs and actin-based ruffles are largely distinct and have unique enrichment profiles for lipids and proteins, RTKs are not passively incorporated into PMμDs. Indeed specific signaling functions of RTKs may remodel certain properties of PMμDs, thus effecting partial overlap of these microdomains. In the absence of EGF stimulation, EGFR is found in lipid microdomains enriched in GM1, and EGF stimulation results in coalescence of these lipid PMμDs with GPIenriched cholesterol microdomains [112]. Interestingly, EGF stimulation also results in the formation of plasma membrane clathrin structures rich in the lipid raft marker GM1 [96]. Interestingly, nearly all EGFR-positive clathrin structures contained GM1, while other clathrin structures were not selectively enriched in GM1. Plasma membrane clathrin structures are indeed highly heterogeneous in size, lifetime, and composition [139,141,143,146,274,275]. Taken together, this suggests that a subset of clathrin structures may be more lipid-ordered and harbor lipid raft components upon EGF stimulation. These studies begin to suggest that a straightforward view of purely independent PMμDs may be too simplistic, and instead suggest that PMμDs form more of a continuum, with highly dynamic, partial spatial overlap of certain component proteins and/or lipids. Many studies to date have examined how a single, particular PMμD controls receptor signaling. While much has been learned from this, systematic study of the dynamic localization of RTKs and activated signaling intermediates within multiple PMμDs at the same time will greatly further our understanding of how PMμDs control receptor signaling. Study of the context-specific signaling of RTKs within specific microdomains will greatly benefit from the continued development of fluorescent signaling biosensors that allow spatiotemporal visualization of individual signaling events within defined PMμDs at high resolution. Given the role that RTKs play in human physiology and the impact that disruption of RTKs has in human diseases such as cancer, it is of great interest to understand the spatiotemporal regulation of RTK signaling within a range of specific PMμDs. Many of the proteins and lipids that underlie the formation of PMμDs are associated with altered RTK signaling and specific diseases in vivo [276]; however, these proteins are also found outside of PMμDs and may have many other possible functions. Future studies that will continue to expand our understanding of how the spatial and temporal organization of receptor signaling controls cellular and systemic physiology will provide crucial information about the context-dependent role of receptor tyrosine kinases in diseases such as cancer and diabetes.

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Acknowledgments We would like to acknowledge the many excellent studies related to control of receptor tyrosine kinase signaling that we were not able to include in this work. C.N.A. is supported by Operating Grant No. 125854 from the Canadian Institutes of Health Research. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20]

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