The Role of the PH Domain and SH3 Binding Domains in Dynamin Function

Abstract

Dynamin, a 100 kD GTPase, is necessary for the normal development and function of mammalian neural tissue. In neurons, it is necessary for the biogenesis of synaptic vesicles, and in other cell types dynamin has a general and important role in clathrin mediated receptor endocytosis. Different isoforms function as molecular scissors either during the formation of coated vesicles from plasma membrane coated pits, or during the release of intracellular vesicles from donor membranes. The mechanism entails the formation of a horseshoe-shaped dynamin polymer at the neck of the budding vesicle, followed by neck scission through a GTP hydrolysis dependent activity. The primary sequence of dynamin contains several C-terminal SH3 binding proline motifs, a central pleckstrin homology (PH) domain, and an N-terminal GTPase domain. Each of these domains appears to play a distinct role in dynamin function. Dynamin is activated by stimulus coupled PKC phosphorylation in brain, possibly mediated through PKC interactions with the PH domain. Further, SH3 domain interactions with the C-terminal sequences and phophatidylinositol/Gβγ interactions with the PH domain also increase dynamin GTPase activity. Each of these various regulatory mechanisms is important in dynamin function during vesicle budding, although the means by which these mechanisms integrate in the overall function of dynamin remains to be elucidated.

Introduction

Many cellular responses to environmental factors are mediated by plasma membrane receptors such as G-protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) [1]. These receptors can activate a variety of intracellular signalling pathways, ultimately resulting in a plethora of cellular responses. For example, activation of RTKs by a number of growth factors (such as EGF, PDGF and NGF) leads to the stimulation of multiple intracellular signalling mechanisms, including the GTPase ras mediated raf/MAP kinase pathway [2]. Similarly GPCRs mediate signal transduction events by activating a variety of signal transducing proteins such as protein kinase A and phospholipase B and C isoforms. These intracellular signalling events in turn activate differentiation pathways or generate mitogenic responses, according to the cell type and/or the receptor involved [3].

Following prolonged or repeated exposure to receptor agonists, signalling mediated through plasma membrane receptors can be attenuated by a variety of mechanisms [4], including internalisation of the receptor by endocytosis 5, 6. Thus, endocytosis has a suppressive effect on membrane receptor signalling. In addition to this function, endocytosis also plays an important role in the determination of cell morphology by regulating the activity of surface receptors that interact with the extracellular matrix [7].

The physiological requirement for endocytosis of plasma membrane components is most readily apparent in neural tissue 8, 9, 10. Endocytosis of the presynaptic plasma membrane components is a necessary step in synaptic vesicle biogenesis (Fig. 1). Endocytosis is therefore critical for synaptic signal transduction, and its suppression in neural tissue can rapidly lead to paralysis.

Endocytosis is initiated by the localised formation of clathrin-based coated pits on the cytoplasmic side of the plasma membrane (Fig. 1). The coated pits, which contain receptors, are internalised and thus released into the cytoplasm by the fission of budding-coated vesicles from the plasma membrane in a process that requires GTP hydrolysis [11]. This stage of vesicle formation appears to require the formation of a protein polymer of dynamin [12]. A short helical coil or horseshoe-shaped structure, principally containing dynamin [13], forms at the neck of budding vesicles. Under normal physiological conditions the activity of this dynamin oligomer is absolutely required for vesicle pinch-off. Dynamin has GTPase activity, and mutations of the GTPase activity are dominant, both suppressing polymer disassembly and completely inhibiting endocytosis 7, 14, 15.

The importance of dynamin-dependent endocytosis in the development and normal function of tissues is underscored by the phenotype of Drosophila dynamin mutants [16]. Shibire, the Drosophila homologue of mammalian dynamin, shares 70% homology with the major mammalian neuronal form, dynamin 1 [17]. In adult flies, temperature sensitive shibire mutations result in rapid paralysis following temperature shift, due to inhibition of synaptic vesicle biogenesis 8, 9, 18. The same shibire mutation also causes severe embryonic deficits in neurogenesis following temperature shift [16]. The mechanism by which this occurs is not clear, but it presumably results from deregulation of growth factor receptors required for neural development and/or perturbation of critical cell adhesion properties.

It appears, therefore, that dynamin is a protein of substantial importance to the control of signal transduction at both the intra- and intercellular levels. Dynamin is recruited to growth factor receptors in response to PDGF, EGF, HGF and insulin 19, 20, 21, where it takes part in the formation of the receptor associated multiprotein complexes required for signal transduction. In the process, it binds to components such as Grb2, PLCγ and the p85 subunit of PI3 kinase 20, 21, 22. Dynamin that has been recruited to the activated receptor must also integrate with the clathrin based multi-protein complex required for coated pit processing so that endocytosis can be properly completed. The molecular events underlying interaction between the two events in which dynamin plays a role, namely, recruitment to the growth factor receptor and integration into the forming coated pit, are not yet clear.

The presence of an N-terminal GTPase domain is readily apparent from the dynamin primary sequence [23]. Proline rich SH3 binding sequences and a pleckstrin homology domain are also evident in the dynamin primary sequence (Fig. 2A). The activity of dynamin appears to be modulated by interaction with signalling molecules such as src homology type-3 (SH3) domain containing proteins and pleckstrin homology (PH) domain ligands. These interactions are the subject of this review.

Protein-protein interactions are often based on the physical association of distinct protein modular domains 24, 25, 26. Domains involved in protein-protein interactions include SH2, SH3, WW, PTB and PH domains. Signal transduction by RTKs, for example, generally involves the interaction of receptor phosphotyrosine residues with distinct domains of downstream signalling proteins, such as Grb2 and PLCγ [2]. Two modular domains that interact with phosphotyrosine residues, SH2 (for src homology type 2) and PTB (for phosphotyrosine binding) mediate these interactions. SH2 domain containing signalling proteins can act as intermediaries in signal transduction by binding to receptor phosphotyrosine residues. Many of the SH2 domain containing proteins also contain another region homologous to a src domain, known as SH3, which also functions as a distinct protein binding site. Grb2, represents an extreme among such proteins, since it is comprised entirely of linker domains. Grb2 links to activated RTKs by interaction of its SH2 domain with receptor phosphotyrosine residues, while also interacting with signalling proteins such as Sos through two flanking SH3 domains. The interaction of SH3 domains with downstream effectors occurs through binding to distinct proline-rich sequences of target molecules [27]. The minimal recognition sequence for binding to an SH3 site has been described as the proline motif sequence PxxP 28, 29. The proline residues are necessary for the formation of a short left-handed polyproline-II helix (PPII), permitting interaction of proline and hydrophobic residues (φ) in a φPxφP sequence with three hydrophobic sites on one side of an SH3 domain [30]. Additional interactions, involving arginine and other residues N-terminal or C-terminal to the PxxP motif, confer specificity of a proline motif sequence for a given SH3 domain 31, 32. The presence of a PxxP motif in a protein sequence therefore suggests the presence of an SH3 domain binding site. The C-terminus of dynamin is very proline rich (32% compared to 4.6% protein average), and contains as many as six PxxP motifs (Fig. 2B). If a requirement for an arginine residue at positions −3 or +5 relative to the PxxP motif is considered, then dynamin contains two separate potential SH3 binding sequences (designated P1 and P2, Fig. 2). In experiments using GST-SH3 domain fusion proteins as probes, dynamin has indeed been shown to be a major SH3 domain binding protein in brain extracts [22]. Dynamin binds tightly to the SH3 domain of several proteins, notably Grb2 and PLCγ. A peptide (P1) corresponding to a proline motif sequence of the dynamin C-terminus (residues ⁷⁷⁸SPTPQRRAPAVPPARPGS⁷⁹⁵) can bind as an independent element to GST-SH3 domain fusion proteins [22]. Binding of dynamin to SH3 proteins therefore most likely occurs by direct interaction of a dynamin C-terminal proline motif sequence with the PPII helix binding surface of SH3 domains. We found that another dynamin C-terminus derived peptide (P2) (residues ⁸²²SPDPFGPPPQVPSRPNR⁸³⁸) also bound SH3 domain proteins [20]. Interestingly, we found that the SH3 domain binding specificities of dynamin peptides P1 and P2 were very different, suggesting that at least two distinct SH3 binding PxxP motif sequences are present in the dynamin C-terminus (discussed in more detail below).

Interaction of dynamin with several SH3 domain proteins in vivo appears likely since immune precipitates with dynamin antibody contain a number of SH3 domain proteins in a variety of cell types 20, 21, 33, 34, 35. Dynamin immune precipitates from such diverse cells as 3T3 mouse fibroblasts, PC12 pheochromocytomas, Chinese hamster ovary cells, human monocytes and Madine-Darby canine kidney (MDCK) cells all demonstrate that the SH3 domain proteins Grb2 and/or PLCγ associate specifically with dynamin. Both Grb2 and PLCγ contain SH2 domains as well, which allow these proteins to bind to phosphotyrosine residues of activated RTK's. It has therefore been proposed that proteins such as Grb2 and PLCγ, by possessing both SH2 and SH3 domains, could thus directly link dynamin to activated RTKs [36](Fig. 3A).

Upon induction of cells with a growth factor such as PDGF, many proteins with SH3 domains can be expected to participate in the growth factor receptor response. We have shown, in immune precipitation experiments, that dynamin is indeed recruited to the tyrosine phosphorylated PDGF receptors after PDGF stimulation [20]. Association of dynamin with other RTKs, such as the EGF and HGF receptors, has similarly been demonstrated [21]. Furthermore, Grb2 has been reported to mediate the growth-factor induced association of a number of receptor-associated proteins, such as the insulin receptor substrate IRS-1 and the focal adhesion kinase p125^FAK, with dynamin 19, 33. Taken together, these results suggest that the recruitment of dynamin to the activated receptor is common and may be functionally significant. A requirement of the dynamin GTPase activity for endocytosis of the transferrin receptor has in fact already been demonstrated 7, 14, 37. In this work, mammalian cells transfected with cDNAs encoding GTPase-defective dynamin exhibited greatly reduced rates of clathrin-coat-mediated transferrin receptor endocytosis.

Dynamin GTPase activity is believed to be necessary for a structural change of the dynamin collar that forms around the neck of budding coated vesicles 13, 38. Although the nature of the structural change in unknown, one can imagine that a GTPase-dependent constriction of the dynamin collar could pinch a budding vesicle off the plasma membrane (Fig. 1). The dynamin super-structure would thus essentially function as a “molecular scissors.” The in vitro interaction of proteins with dynamin through their SH3 domains activates its GTPase activity 22, 39. Binding of dynamin to proteins containing SH3 domains, such as PLCγ or Grb2, that in turn associate with activated receptor tyrosine kinases, could hence thereby couple receptors with the machinery necessary for endocytosis. Such association could thus lead to dynamin GTPase induced internalisation and down-regulation of the receptor, induced by the GTPase activity of dynamin helicoı̈dal oligomers. Indeed, recent microinjection experiments have demonstrated that Grb2 is required for endocytosis of the EGF receptor, and that this requirement specifically involves the C-terminal Grb2 SH3 domain [35].

Several dynamin isoforms (dyn-1,dyn-2 and dyn-T) have been identified 40, 41, 42, 43. Dynamin-1 and dynamin-2 isoforms, which are coded by separate genes, have recently been demonstrated to be functionally different 44, 45. While dynamin-1 is clearly involved in early stages of endocytosis at the plasma membrane, dynamin-2 appears to be associated with Golgi membranes. Dynamin-1 and dynamin-2 isoforms therefore probably do not bind to the same membrane associated proteins. The N-terminal GTPase domain sequences of the dynamin isoforms have a high degree of homology (88% sequence homology between dynamin-1 and dynamin-2 isoforms). In contrast, although the C-terminal sequences of the isoforms are both proline rich, they differ considerably in sequence (only 53% homology between dynamin-1 and dynamin-2 C-termini). These differences in the SH3 domain binding sequences may reflect a need for specific SH3 interactions.

The dynamin-1 isoform is particularly abundant in brain 41, 46, 47where it may interact with SH3 domain proteins specific to neural tissue. For example, amphiphysin, an integral membrane protein of synaptic vesicle membranes [48], specifically associates with dynamin in immune-precipitates from synaptosomal fractions [49]. The interaction of amphiphysin with dynamin appears to occur through its SH3 domains and appears to be very specific, since dynamin is essentially the only protein in brain lysates retained on an amphiphysin SH3 domain affinity matrix. the relevance of this interaction for synaptic functioning is presently not clear, although in light of the effect of SH3 domain binding on dynamin GTPase activity, the interaction with amphiphysin is probably necessary for effective dynamin “molecular scissors” activity at the presynaptic nerve terminal [50].

Interaction with SH3 domains occurs by binding to proline motif sequences in the dynamin C-terminus. The presence of multiple proline motifs in SH3 binding proteins is common and also occurs, for example, in SOS and Cbl 51, 52. We have tested whether these multiple PxxP motifs represent a redundancy of binding sites, or whether they, in fact, function as distinct and separate SH3 domain binding sites within a single polypeptide chain. Using matrix coupled peptides corresponding to several PxxP motifs of dynamin and Sos, we probed for binding to proteins in cell lysates. We found that each PxxP motif peptide tested bound a unique subset of SH3 domain proteins in cell lysates [20]. The multiple PxxP motifs in dynamin, and in Sos, therefore probably represent separate and distinct SH3 binding sites. Dynamin and Sos both appear to be generally implicated in RTK-related events. A specific interaction between dynamin and a variety of SH3 proteins that associate with various RTKs can hence be achieved through binding to multiple specific SH3 binding sites within a single polypeptide chain. The presence of multiple specific binding sites, rather than a single non-specific SH3 binding site, allows for multiple specific interactions with SH3 domain proteins.

The cytoskeletal protein pleckstrin is the major protein kinase C substrate in platelets [53]. The N- and C-terminal domains of pleckstrin, each of 100 amino-acid residues in length, have similar amino-acid sequences. Each of these domains is believed to function in protein-protein and protein-lipid interactions, and represents the prototype for what has come to be known as the pleckstrin homology (PH) domain 54, 55. An increasing number of proteins that are important for signal transduction and cytoskeletal function, including dynamin, have been found to contain sequences related to those of the pleckstrin PH domains 54, 56, 57. Although the primary sequence homology among pleckstrin homology domains is typically very limited (generally 21–25% identity), the three dimensional structures of the PH domains of several proteins show a striking degree of structural similarity 58, 59, 60, 61, 62, 63. The PH domains of dynamin, spectrin and pleckstrin are all composed of orthogonal β sheets around a central hydrophobic cleft and α helix. This structure is related to those of several lipid binding proteins such as lipocalins and retinol binding protein (RBP) [62], as well as the recently identified phosphotyrosine binding domain (PTB) [64].

Prior to the determination of the PH domain structure, segments of synapsin I, spectrin, beta-adrenergic receptor kinase (β-ARK), PLCγ and PLCδ, that are now known to contain PH domains, were identified as membrane and/or phospholipid binding sites 65, 66. Specific binding of inositol phospholipids to bacterially expressed PH domains of pleckstrin, ras-GAP, Tsk, β-ARK and PLCγ has been demonstrated using a centrifugal assay [67]. Further, positively charged residues at the lip of the β-barrel were identified as sites for interaction with phosphatidylinositols by determination of NMR chemical shift changes upon binding of ptdIns 4, 5P₂ to the pleckstrin PH domain. The recently obtained structures of phosphoinositol associated spectrin and pleckstrin PH domains demonstrate that inositol phosphate moieties interact with charged residues at the edge of the cleft 68, 69. Lipid fatty-acid moieties of phosphatidylinositols probably fit into the hydrophobic cleft of the PH domain.

The binding of dynamin-1 to membranes and lipids might be expected in light of the well established function of dynamin-1 in endocytic vesicle formation. As we and others have demonstrated, dynamin is associated with cell membranes 46, 70. Much of the dynamin in extracts of brain tissue occurs in particulate fractions, and, further, it can be isolated from purified synaptosomal membranes [46]. Additionally, the dynamin-2 isoform, which also has a pleckstrin homology domain, has recently been isolated in association with Golgi membrane fractions from liver tissue [44]. The interaction with membranes appears to be due to a direct interaction with lipids, since purified dynamin has been shown to bind directly to phospholipid coated glass beads [71]and, further, phospholipids and membrane vesicles stimulate the dynamin GTPase activity [70].

The interaction of dynamin with, and its activation by, specific phospholipids (such as phosphatidylinositols) probably occurs through its PH domain since a dynamin PH domain GST fusion protein binds phospholipids specifically 72, 73. Further, it has recently been demonstrated that deletion of the dynamin PH domain abolishes activation of its GTPase activity by phosphatidylinositol [73].

Dynamin appears to have multiple binding sites that are capable of interacting with membrane components. In addition to lipid binding by the PH domain, dynamin has been shown to interact in vitro with the vesicle coat component β-adaptin [74]. The PH domain and the β-adaptin binding sites may act synergistically to generate a very high affinity interaction with membranes. Although these in vitro interactions have been well documented, it should be noted that these two distinct interactions of dynamin with membrane components have yet to be demonstrated to occur in vivo.

Dynamin phosphorylation may play a role in synaptic transmission since cycles of synaptosome depolarisation and repolarisation result in a cycle of dynamin dephosphorylation and rephosphorylation [75]. It appears that dynamin is dephosphorylated by calcineurin upon depolarisation of pre-synaptic terminal plasma membranes [76], while PKC rephosphorylates dynamin following neurotransmitter release. PKC specifically phosphorylates the dynamin-1 isoform in vitro (resulting in a 12-fold activation of the dynamin GTPase activity) [77]. In addition to dynamin, a number of other PH domain proteins (such as pleckstrin, Bruton's tyrosine kinase (Btk), beta- adrenergic receptor kinase (β-ARK) and Akt/rac-kinase) are PKC substrates and appear to be regulated by PKC mediated phosphorylation 4, 78, 79, 80. In the case of Btk, interaction with PKC appears to involve direct association with the PH domain [80]. It will be interesting to determine whether a similar interaction of PKC with the PH domain of dynamin is required for regulation of synaptic vesicle biogenesis by dynamin phosphorylation.

PH domains have also been shown to bind the βγ subunits of heterotrimeric G-proteins 81, 82, 83, 84, 85. The interaction with G_βγ is particularly strong for the PH domain of β-ARK, a kinase which attenuates beta-adrenergic receptor signalling by phosphorylation of the receptor. The binding of β-ARK to G_βγ apparently serves to target this kinase to the G-protein associated membrane bound receptor [86]. Recently, it has been reported that G_βγ subunits can activate the GTPase activity of dynamin [87]. It will be of interest to determine whether interaction of dynamin with GPCR associated G_βγ subunits may link dynamin to these receptors, as may be the case for Grb2 with RTKs and integrin receptors (Fig. 3).