**2.3 Profilin ligands**

Despite its relatively small size, many profilin ligands have by now been recognized, such as actin and actin-related proteins, polyphosphoinositides, PLP, annexin-I, and the list still increasing (Schlüter et al., 1997). Recently, there are more than 50 described profilin-binding

In this context, Gieselmann et al., (1995) showed that human profilin-I exhibits about five folds higher affinity for actin than profilin-II. Radiography analyses of the structures of human profilin isoforms imply that the substitution of profilin-I S29 by Y29 in profilin-II participates in the higher affinity of profilin-II for proline-rich sequences (Nodelman et al., 1999). In spite of the similarity in the 3D structures of human profilin-I and -II, the surface characteristics, such as exposure of hydrophobic patches (Figure 2), and biochemical

Fig. 1. Profilin-I isoforms from different organisms showing a similar helix (red) and strand (cyan) structure (PDB database: 1PFL, 1KOK, 2PRF, and 3NUL) with the loops highlighted in

Despite its relatively small size, many profilin ligands have by now been recognized, such as actin and actin-related proteins, polyphosphoinositides, PLP, annexin-I, and the list still increasing (Schlüter et al., 1997). Recently, there are more than 50 described profilin-binding

green, adapted from Krishnan & Moens, (2009) with permission.

**2.3 Profilin ligands** 

properties of each isoform are different (Krishnan & Moens, 2009).

ligands from diverse origins. However, this represents only a part of the real number of profilin-binding partners. Figure 3 shows the identified profilin ligands in mammalian cells. These do not include only molecules of focal contacts that could link profilin directly to actin polymerization such as VASP (vasodilator-stimulated Phosphoprotein) or Mena (mouse homolog of Drosophila enabled) (Gertler et al., 1996; Parast & Otey, 2000; Reinhard et al., 1995) but also include other molecules such as nuclear-export receptors (Boettner et al., 2000; Camera et al., 2003), regulators of endocytosis and membrane trafficking (Witke et al., 1998), Rac and Rho effectors molecules (Alvarez-Martinez et al., 1996; Miki et al., 1998; Ramesh et al., 1997; Suetsugu et al., 1998; Watanabe et al., 1997; Witke et al., 1998; Yayoshi-Yamamoto et al., 2000) and synaptic scaffold proteins (Mammoto et al., 1998; Miyagi et al., 2002; Wang et al., 1999). While a small number of these interactions demonstrated a physiological relevance, the recognition of profilin-interacting proteins could explain the unpredicted roles of profilin in mammalian cells. The profilin-ligands binding might help in linking different pathways to cytoskeletal dynamics via a mechanism that still unknown. Instead, the profilin–ligand interaction might work independently of actin to control the ligands directly (Witke, 2004).

Fig. 2. Structure of human profilin-I and –II: differences in the surface-charge distribution might account for the ligand-binding specificity of profilin-I and -II. Colored regions highlight amino acid residues that are different in profilin-I and -II. Non-conserved residues are shown in blue; conserved residues are shown in brown, adapted from Witke, (2004) with permission.

Among this large number of profilin ligands we will focus on the binding of profilin to some of those ligands believed to be of relevant role in vascular problems such as actin and ligands in Rho/Rac pathway.

#### **2.3.1 Profilin, actin & cytoskeleton**

*In vitro*, Profilins can interact with and sequester actin monomers, in that way diminishing the concentration of free actin monomers that are accessible for filament elongation (Carlsson et al., 1977). They refill the pool of ATP-actin monomers via rising the nucleotide exchange rate by 1000-fold in comparison with that rate obtained from simple diffusion (Goldschmidt-Clermont et al., 1992). The profilin–ATP–actin complex can bind to the fast growing, barbed, or plus end of the actin filament and liberate the ATP–actin monomer, which is after that added to the filament (Figure 4). As a result, the elongating filament is made of ATP-actin. Down the filament, the ATP is slowly hydrolyzed via the actin intrinsic

ATPase activity. This produces ADP–actin in the older part of the filament. ADP–actin can be liberated gradually from the pointed or minus end of the filament by depolymerization or at faster rate by actin-depolymerizing proteins (Witke, 2004).

Fig. 3. Network of molecular interactions of profilin. Proteins that are known to interact with profilin are grouped according to their cellular location or the complexes in which they are found. Some of the profilin ligands are shared among different complexes (indicated by the intersecting fields), which suggests a crosstalk among signaling platforms, with profilin as the common denominator. Several links exist to small GTPases such as Rac1, RhoA, cdc42, Ras and Rap that are part of pathways that signal to the actin cytoskeleton. For simplicity, the term profilin is commonly used for profilin-I and profilin-II. Direct interactions between profilin and the ligands are indicated by unbroken lines, whereas potentially direct interactions are indicated by broken lines. Abbreviations: AF-6, All-1 fusion partner from chromosome 6; EVL, Ena VASP like; FMRP, fragile X mental retardation protein; FRL, formin-related gene in leukocytes; HSP, heat-shock protein; Mena, mouse homolog of Drosophila enabled; POP, partner of profilin; SMN, survival of motor neuron; VASP, vasodilator-stimulated phosphoprotein; VCP, valosine-containing protein; WASP, Wiskott– Aldrich syndrome protein; WAVE, WASP family verprolin-homologous protein; WIP, WASP-interacting protein, adapted from Witke, (2004) with permission.

It is worth noting that the presence of other G-actin binding proteins, such as thymosin β4 or any of the ADF family members can alter these processes (Pantaloni & Carlier, 1993). Additionally, capping the plus end of the filaments inhibits the addition of the profilin-actin complexes and consequently limits the activity of profilin to a simple sequestering effect (Pantaloni & Carlier, 1993; Perelroizen et al., 1996; Pring et al., 1992). Thus, the presence of other G-actin binding and/or capping proteins could regulate the profilin effect on cellular actin (Schlüter et al., 1997).

ATPase activity. This produces ADP–actin in the older part of the filament. ADP–actin can be liberated gradually from the pointed or minus end of the filament by depolymerization

Fig. 3. Network of molecular interactions of profilin. Proteins that are known to interact with profilin are grouped according to their cellular location or the complexes in which they are found. Some of the profilin ligands are shared among different complexes (indicated by the intersecting fields), which suggests a crosstalk among signaling platforms, with profilin as the common denominator. Several links exist to small GTPases such as Rac1, RhoA, cdc42, Ras and Rap that are part of pathways that signal to the actin cytoskeleton. For simplicity, the term profilin is commonly used for profilin-I and profilin-II. Direct interactions between

It is worth noting that the presence of other G-actin binding proteins, such as thymosin β4 or any of the ADF family members can alter these processes (Pantaloni & Carlier, 1993). Additionally, capping the plus end of the filaments inhibits the addition of the profilin-actin complexes and consequently limits the activity of profilin to a simple sequestering effect (Pantaloni & Carlier, 1993; Perelroizen et al., 1996; Pring et al., 1992). Thus, the presence of other G-actin binding and/or capping proteins could regulate the profilin effect on cellular

profilin and the ligands are indicated by unbroken lines, whereas potentially direct interactions are indicated by broken lines. Abbreviations: AF-6, All-1 fusion partner from chromosome 6; EVL, Ena VASP like; FMRP, fragile X mental retardation protein; FRL, formin-related gene in leukocytes; HSP, heat-shock protein; Mena, mouse homolog of Drosophila enabled; POP, partner of profilin; SMN, survival of motor neuron; VASP, vasodilator-stimulated phosphoprotein; VCP, valosine-containing protein; WASP, Wiskott– Aldrich syndrome protein; WAVE, WASP family verprolin-homologous protein; WIP,

WASP-interacting protein, adapted from Witke, (2004) with permission.

actin (Schlüter et al., 1997).

or at faster rate by actin-depolymerizing proteins (Witke, 2004).

Fig. 4. Role of profilins in actin polymerization. An actin filament consists of two α-helical protofilaments. *In vitro*, three major functions have been identified for profilins in the regulation of actin polymerization. (i) Profilins can bind to and sequester actin monomers, thereby decreasing the concentration of free actin monomers that are available for filament elongation. (ii) Profilins replenish the pool of ATP–actin monomers (red) by increasing the rate of nucleotide exchange on the bound actin monomer 1000-fold compared with the rate of exchange based on simple diffusion. (iii) The profilin–ATP–actin complex can interact with the fast growing end of the actin filament and release the ATP–actin monomer, which is then added to the filament. Consequently, the elongating filament consists of ATP–actin. Along the filament, the ATP is slowly hydrolyzed by the intrinsic ATPase activity of actin, which generates ADP–actin (orange) in the older part of the filament. ADP–actin can be released slowly from the end of the filament by depolymerization or at an accelerated rate by 'actin-depolymerizing proteins' (not shown), adapted from Witke, (2004) with permission.

Previously, it has been considered that the profilins effect on nucleotide exchange on actin directly regulates their ability to promote filament assembly at the plus end. Polymerization of filament is coupled with the actin-bound ATP hydrolysis and thus far, profilins are unique microfilament associated proteins that can work as nucleotide exchange factors. Polymerization of ATP-actin occurs more rapidly and at a lower critical concentration than ADP-actin (Pollard, 1986). Nonetheless, profilin isoforms I and III in *Arabidopsis* are unable to speed up the rate of nucleotide exchange on G-actin yet still reduce the critical concentration at the plus ends of filaments, similar to vertebrate profilin (Perelroizen et al., 1996). These data demonstrate that the major effect of profilins on actin polymerization cannot be linked with their capacity to work as nucleotide exchange factors.

*In vivo*, the global view that the main biological function of profilin was observed in its actin sequestering effect became debatable, principally due to the finding that the concentration of profilin in cells and its actin-binding affinity are inadequate to stabilize the G-actin pool (Babcock & Rubenstein 1993; Goldschmidt-Clermont et al., 1991; Machesky & Pollard, 1993; Sohn & Goldschmidt-Clermont, 1994). Generally, the data obtained from cells with different profilin levels are in harmony with the notion that in lower eukaryotes the central role of profilin is to sequester G-actin, whereas in higher eukaryotes this is mainly done via other G-actin binding proteins such as thymosin β4 (Safer et al., 1991), and profilins are mostly implicated in the actin filament dynamics control (Sohn & Goldschmidt-Clermont, 1994).

Based on this notion, lower eukaryotes deficient in profilins should exhibit an increase in Factin, however in higher eukaryotes this would not be the principal outcome. Compatible with this model, *S. pombe* cells with profilin overproduction showed undetectable amount of actin filaments, and are incapable of forming a contractile ring (Balasubramanian et al., 1994). In *S. cerevisiae* cells harmful effects due to actin overexpression, could be compensated by profilin overexpression (Magdolen et al., 1993). Conversely, several studies reported about filament-stabilizing or -regulating functions of profilin in higher eukaryotes. For example, the overall F-actin content and stability were elevated whereas; a considerable amount of F-actin was shifted from stress fibers to the cortical actin network in Chinese hamster ovary cells overexpressing profilin (Finkel et al., 1994). Likewise, actin filaments were stabilized against cytochalasin D and latrunculin in baby hamster kidney cells overexpressing birch profilin (Rothkegel et al., 1996). In addition, a shift in F-actin from stress fibers to thick peripheral actin filament bundles with a corresponding increase in cellular adhesion to fibronectin has been reported in cultured human endothelial cells overexpressing profilin (Moldovan et al., 1997).

Although these findings indicated a differential role of profilins between lower and higher eukaryotes, a few studies showed contradictory data to these reports (Cao et al., 1992; Edwards et al., 1994; Staiger et al., 1994). Consequently, a final conclusion on the validity of the assumption regarding differential functions of profilins in higher and lower eukaryotes needs to be confirmed with further experimentations (Schlüter et al., 1997).

#### **2.3.2 Profilin & Rho/Rac pathway**

Rho/Rac signaling pathway represents one of the well-known pathways in the regulation of actin cytoskeleton, as indicated by the Rac1-dependent membrane ruffling and RhoAstimulated stress-fiber formation (Nobes & Hall, 1995). Although there is no any report about the direct interaction between profilins and Rho and/or Rac or any other small GTPases, many of the profilin ligands are well-recognized Rho/Rac effector molecules (Witke, 2004). In this regard, our recent data showed that profilin overexpression in vascular smooth muscle cells (VSMC) of transgenic mice results in vascular remodeling and hypertension. These were associated with increased Rho-GTPase activity and Rhodependent coiled-coil kinase (ROCK) expression (Hassona et al., 2010; Moustafa-Bayoumi et al., 2007). As well, it has been reported that ROCK is a part of the profilin-II complex in the brain (Witke et al., 1998) and this binding is significant in the regulation of neurite outgrowth by ROCK (Da Silva et al., 2003). Furthermore, two other proteins that connect profilin to the Rac pathway were recognized in the profilin-II complex in the brain, Nckassociated protein (Nap 1) and partner of profilin (POP)-130 (Witke et al., 1998). GTP–Rac1

*In vivo*, the global view that the main biological function of profilin was observed in its actin sequestering effect became debatable, principally due to the finding that the concentration of profilin in cells and its actin-binding affinity are inadequate to stabilize the G-actin pool (Babcock & Rubenstein 1993; Goldschmidt-Clermont et al., 1991; Machesky & Pollard, 1993; Sohn & Goldschmidt-Clermont, 1994). Generally, the data obtained from cells with different profilin levels are in harmony with the notion that in lower eukaryotes the central role of profilin is to sequester G-actin, whereas in higher eukaryotes this is mainly done via other G-actin binding proteins such as thymosin β4 (Safer et al., 1991), and profilins are mostly implicated in the actin filament dynamics control (Sohn & Goldschmidt-Clermont, 1994). Based on this notion, lower eukaryotes deficient in profilins should exhibit an increase in Factin, however in higher eukaryotes this would not be the principal outcome. Compatible with this model, *S. pombe* cells with profilin overproduction showed undetectable amount of actin filaments, and are incapable of forming a contractile ring (Balasubramanian et al., 1994). In *S. cerevisiae* cells harmful effects due to actin overexpression, could be compensated by profilin overexpression (Magdolen et al., 1993). Conversely, several studies reported about filament-stabilizing or -regulating functions of profilin in higher eukaryotes. For example, the overall F-actin content and stability were elevated whereas; a considerable amount of F-actin was shifted from stress fibers to the cortical actin network in Chinese hamster ovary cells overexpressing profilin (Finkel et al., 1994). Likewise, actin filaments were stabilized against cytochalasin D and latrunculin in baby hamster kidney cells overexpressing birch profilin (Rothkegel et al., 1996). In addition, a shift in F-actin from stress fibers to thick peripheral actin filament bundles with a corresponding increase in cellular adhesion to fibronectin has been reported in cultured human endothelial cells

Although these findings indicated a differential role of profilins between lower and higher eukaryotes, a few studies showed contradictory data to these reports (Cao et al., 1992; Edwards et al., 1994; Staiger et al., 1994). Consequently, a final conclusion on the validity of the assumption regarding differential functions of profilins in higher and lower eukaryotes

Rho/Rac signaling pathway represents one of the well-known pathways in the regulation of actin cytoskeleton, as indicated by the Rac1-dependent membrane ruffling and RhoAstimulated stress-fiber formation (Nobes & Hall, 1995). Although there is no any report about the direct interaction between profilins and Rho and/or Rac or any other small GTPases, many of the profilin ligands are well-recognized Rho/Rac effector molecules (Witke, 2004). In this regard, our recent data showed that profilin overexpression in vascular smooth muscle cells (VSMC) of transgenic mice results in vascular remodeling and hypertension. These were associated with increased Rho-GTPase activity and Rhodependent coiled-coil kinase (ROCK) expression (Hassona et al., 2010; Moustafa-Bayoumi et al., 2007). As well, it has been reported that ROCK is a part of the profilin-II complex in the brain (Witke et al., 1998) and this binding is significant in the regulation of neurite outgrowth by ROCK (Da Silva et al., 2003). Furthermore, two other proteins that connect profilin to the Rac pathway were recognized in the profilin-II complex in the brain, Nckassociated protein (Nap 1) and partner of profilin (POP)-130 (Witke et al., 1998). GTP–Rac1

needs to be confirmed with further experimentations (Schlüter et al., 1997).

overexpressing profilin (Moldovan et al., 1997).

**2.3.2 Profilin & Rho/Rac pathway** 

interacts with POP-130 and can detach the tetrameric WAVE 1 [Wiskott–Aldrich syndrome protein (WASP) family verprolin-homologous protein]1 complex, resulting in the activation of actin polymerization by WAVE1. Yet, the role of profilin binding to POP-130 is not apparent however it is possible that profilin might manage the complex formation between WAVE1 and POP-130 and between FMRP (fragile X mental retardation protein) and POP-130, in the same way as Rac1 (Witke, 2004).

Additional small-GTPase-binding molecules that can interact with profilin are the Rhobinding molecules, mouse homologs of the *Drosophila* gene diaphanous (mDia1, mDia2 and mDia3) which are known as potent nucleators of actin polymerization (Wallar & Alberts, 2003). Generally, the diaphanous protein exists in an inactive conformation due to folding back of its N terminal GTPase-binding domain onto its C-terminal Dia-autoregulatory domain resulting in association and autoinhibition. RhoA binding to the N terminus releases the autoinhibition and activates actin nucleation (Alberts, 2002). Profilin binding occurs through the proline-rich formin homology domain that present in the core of that diaphanous molecule (Watanabe et al., 1997). Yet, the significance of that binding is not clear. One interesting possibility is that diaphanous can move actin after it has been sequestered by profilin and activate actin polymerization (Li & Higgs, 2003). However, this is limited by the argument that the studies of profilin-diaphanous binding used truncated versions of diaphanous, rather than the full-length protein. *In vivo*, it has been suggested that large complex of diaphanous oligomers is present as well (Li & Higgs, 2003), which via diaphanous monomers can interact with profilin and/or profilactin molecules. Nevertheless, the structure and regulation of this enormous signaling platform for actin nucleation need to be understood (Witke, 2004).

#### **2.3.3 Ligands binding sites**

In this section we will discuss the binding sites of the main profilin ligands, actin, phatidylinositol 4,5-bisphosphate (PtdIns 4,5-P), and PLP. Initially, profilin binds with high affinity (micromolar range) to G-actin in a 1:1 stoichiometric complex (Schlüter et al., 1997). The amino acid motif LADYL in the C-terminal α-helix was first proposed to be implicated in actin binding depending on (1) the presence of this motif in most of profilins, (2) the presence of homologous sequences in a range of actin-binding proteins such as DNase I, fragmin, gelsolin, severin, villin and the vitamin D-binding protein (Binette et al., 1990; Tellam et al., 1989; Vandekerckhove, 1989; Vinson et al., 1993). Nevertheless, this hypothesis was neglected due to (1) the absence of this sequence in mammalian profilins, (2) the ability of Saccharomyces profilin to interact with actin even after deletion of this motif (Haarer et al., 1993). Now, the LADYL-motif is believed to be a central element in the dense structure of these proteins (Ampe & Vandekerckhove, 1994; Fedorov et al., 1994; Haarer et al., 1993; McLaughlin & Weeds, 1995). Studies on bovine profilin-I and β-actin showed that the actin binding sites on profilin are localized in the α-helix 3, the proximal part of α-helix 4, and in the β-strands 4, 5 and 6 (Schutt et al., 1993) (Figure 5). These residues bind to subdomains 1 and 3 on the actin molecule; however, they do not exhibit a conserved sequence motif (Thorn et al., 1997). In the bovine complex, Phe375 appears to be a key residue that interacts with Ile73, His119, Gly121 and Asn124 on the profilin side (Schutt et al., 1993). Similarly, other studies on *Acanthamoeba* reported that actin-related proteins such as Arp2 interact with profilin using the same binding site (Kelleher et al., 1995; Machesky, 1997).

Fig. 5. Topographical relation of the main ligand binding domains as seen on the X-ray structure of bovine profilin (Schutt et al., 1993). The binding domains of actin and actin related proteins (blue; Schutt et al., 1993) and PtdIns 4,5-P2 (red; Sohn et al., 1995) overlap, while that for proline-cluster sequences (green; Metzler et al., 1994) is located at the opposite side of the profilin molecule, adapted from Schlüter et al., (1997) with permission.

On the other hand, studies on *Acanthamoeba* described a positively charged area opposing both termini, placed in the G-actin binding site as the binding motif of the second key ligand of profilin, PtdIns 4,5-P2 (Fedorov et al., 1994) (Figure 5). This was supported by mutation studies on *Saccharomyces* profilin and human profilin-I. Point mutations in this region diminished the binding affinity of profilin to PtdIns 4,5-P2 (Haarer et al., 1993; Sohn et al., 1995). In line with the observation that the binding sites of G-actin and PtdIns 4,5-P2 on profilin overlap (Figure 5), it has been reported that these ligands compete with each other for binding to profilin (Lassing & Lindberg, 1985; 1988; Machesky et al., 1990). In addition, other reports showed that binding of PtdIns 4,5-P2 results in a conformational change in profilin and disrupts the profilin-actin complex (Raghunathan et al., 1992). Also, it has been revealed that profilin can bind a variety of phosphatidylinositol and the binding affinity of human profilin-I to phosphatidylinositol 3,4-bisphosphate (PtdIns 3,4-P2), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns 3,4,5-P3) is higher than its affinity to PtdIns 4,5-P2 (Lu et al., 1996). Furthermore, phosphoinositide (PI) 3-kinase activity may be regulated by profilin through direct binding to the p85 subunit of this enzyme (Singh et al., 1996). PI 3-kinase has no effect on the binding of actin to profilin (Singh et al., 1996), signifying that the binding sites of actin and p85 on profilin are different.

Conversely, the profilin-PtdIns 4,5-P2 complex can be hydrolyzed only via phospholipase Cγ1 (PLCγ1). Phosphorylation and activation of this lipase as a result of transmembrane signaling (Goldschmidt-Clermont et al., 1990; 1991) leads to the conclusions that (1) profilins

Fig. 5. Topographical relation of the main ligand binding domains as seen on the X-ray structure of bovine profilin (Schutt et al., 1993). The binding domains of actin and actin related proteins (blue; Schutt et al., 1993) and PtdIns 4,5-P2 (red; Sohn et al., 1995) overlap, while that for proline-cluster sequences (green; Metzler et al., 1994) is located at the opposite

On the other hand, studies on *Acanthamoeba* described a positively charged area opposing both termini, placed in the G-actin binding site as the binding motif of the second key ligand of profilin, PtdIns 4,5-P2 (Fedorov et al., 1994) (Figure 5). This was supported by mutation studies on *Saccharomyces* profilin and human profilin-I. Point mutations in this region diminished the binding affinity of profilin to PtdIns 4,5-P2 (Haarer et al., 1993; Sohn et al., 1995). In line with the observation that the binding sites of G-actin and PtdIns 4,5-P2 on profilin overlap (Figure 5), it has been reported that these ligands compete with each other for binding to profilin (Lassing & Lindberg, 1985; 1988; Machesky et al., 1990). In addition, other reports showed that binding of PtdIns 4,5-P2 results in a conformational change in profilin and disrupts the profilin-actin complex (Raghunathan et al., 1992). Also, it has been revealed that profilin can bind a variety of phosphatidylinositol and the binding affinity of human profilin-I to phosphatidylinositol 3,4-bisphosphate (PtdIns 3,4-P2), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns 3,4,5-P3) is higher than its affinity to PtdIns 4,5-P2 (Lu et al., 1996). Furthermore, phosphoinositide (PI) 3-kinase activity may be regulated by profilin through direct binding to the p85 subunit of this enzyme (Singh et al., 1996). PI 3-kinase has no effect on the binding of actin to profilin (Singh et al., 1996),

Conversely, the profilin-PtdIns 4,5-P2 complex can be hydrolyzed only via phospholipase Cγ1 (PLCγ1). Phosphorylation and activation of this lipase as a result of transmembrane signaling (Goldschmidt-Clermont et al., 1990; 1991) leads to the conclusions that (1) profilins

side of the profilin molecule, adapted from Schlüter et al., (1997) with permission.

signifying that the binding sites of actin and p85 on profilin are different.

are implicated in the metabolism of phosphoinositide and (2) hydrolysis of PtdIns 4,5-P2 causes profilin to move out from the membrane to the cytosol where it can bind to actin or other ligands. These conclusions propose that profilin–phosphoinositide binding plays a vital role *in vivo* (Janmey et al., 1995; Ostrander et al., 1995).

Mutation (Björkegren et al., 1993; Haarer et al., 1993) and NMR (Archer et al., 1994; Metzler et al., 1994) analyses described the binding site of profilin to the third main ligand, PLP as a hydrophobic patch including the NH- and COOH-terminal α-helices and the upper face of the antiparallel β-sheet, opposing to the actin/PtdIns 4,5-P2 binding region (Figure 5). The binding of PLP to profilins has no effect on the interaction with Gactin or PtdIns 4,5-P2 (Archer et al., 1994; Kaiser et al., 1989), indicating that PLP has a distinct binding site (Figure 4). Expediently, this specific PLP-profilin binding is used for profilins purification (Kaiser et al., 1989; Lindberg et al., 1988). For effective profilin binding, it has been proposed that 6 continuous prolines would be sufficient (Metzler et al., 1994). Nevertheless, other reports demonstrated that at least 8–10 prolines are required for efficient binding (Domke et al., 1997; Machesky & Pollard, 1993; Perelroizen et al., 1994; Petrella et al., 1996).These proline stretches may be interrupted by single glycine residues (Domke et al., 1997; Lambrechts et al., 1997) and may be capable of simultaneous binding of two profilins (Lambrechts et al., 1997), depending on the ability of profilin to oligomerize (Babich et al., 1996).

The first recognized ligand for PLP was VASP, a focal adhesion molecule that was reported to interact directly with F-actin (Jockusch et al., 1995; Reinhard et al., 1995), and it also described as a substrate of both cGMP- and cAMP-dependent protein kinases in platelets (Halbrügge et al., 1990). VASP has a central proline-rich domain with a single copy and a 3 fold tandem repeat of a remarkable (G)P5 motif (Haffner et al., 1995). This motif is both required and sufficient for profilin binding (Domke et al., 1997; Lambrechts et al., 1997; Reinhard et al., 1995). Another PLP-binding ligand similar to VASP is a VASP-related mouse protein, Mena (Gertler et al., 1996). Additional PLP-biding ligands are the formin-related proteins, *S. cerevisiae* Bni1p and Bnr1p, *S. pombe* Cdc12p, *Drosophila* cappuccino and p140mDia, the mammalian homologue of the *Drosophila* protein diaphanous (Chang et al., 1997; Evangelista et al., 1997; Imamura et al., 1997; Manseau et al., 1996; Watanabe et al., 1997). These proteins have a proline-rich domain with numerous proline stretches consisting of 5–13 residues and a C-terminal consensus sequence of approximately 100 amino acids (Castrillon & Wasserman, 1994). Due to the high specific binding of Bni1p, Bnr1p and p140mDia to the GTP-bound form of Rho family members (Kohno et al., 1996; Imamura et al., 1997; Watanabe et al., 1997); they perhaps represent significant connectors between signal transduction, profilin and the cytoskeleton. Furthermore, adenylyl cyclase-associated protein (CAP) has been described as PLP-binding ligand. CAP has a G(P)6 G(P)5 motif and it can bind to profilin (Domke et al., 1997; Lambrechts et al., 1997). Nevertheless, other studies demonstrated that CAP exists in a folded configuration (Lambrechts et al., 1997) and hence its binding to profilin may be firmly regulated.

#### **2.3.4 Regulation of profilin-ligands binding**

The important factors that could help in understanding the process of profilin-ligand binding regulation include the structural requirements for the binding of profilin to the ligand, the binding specificity of ligands to different profilins and the mechanisms of ligand release. Initially, the structural requirements for the profilin-ligand binding are not completely understood. In spite of binding of profilin to an extremely diverse group of ligands either directly or as part of a larger complex, the binding sites on both profilin and ligands appear to be well conserved. The majority of ligands are believed to interact with the PLP domain of profilin that contains the N- and C-terminal helices. The only exception, so far, to this model is gephyrin, which appears to bind to a special profilin domain (Giesemann et al., 2003). All profilin ligands are characterized by the presence of stretched or nearly stretched proline-rich domains that are required for profilin binding. Still, a contiguous prolines stretch is insufficient. Depending on the data obtained from *in vitro* studies using synthetic PLP peptides of different length high-affinity binding requires a decamer as a minimum, (Perelroizen et al., 1994) however this cannot be extended to cover proteins or to be used for recognizing or evaluating the ability of profilin to bind to a ligand. A lot of profilin ligands contain in their proline-rich domains proline repeats of no more than three or four successive prolines. Further amino acids, mostly glycines, appear to be capable of replacing proline, and an efficient profilin-binding domain appears to include numerous repeats that have the consensus sequence ZPPX (where Z=P, G or A; and X= any hydrophobic amino acid) (Witke et al., 1998).

The second important factor in regulating the binding of profilins to their ligands is the binding specificity of ligands to the different profilins. Previous reports showed that the interaction of ligands with profilin-I and profilin-II occurs in a highly specific manner (Witke et al., 1998) and it looks likely that it is not only the PLP-binding domains but also other complex binding parameters have to be considered. Comparative studies on the structures of mammalian profilin-I and profilin-II indicated that they are approximately superimposable (Nodelman et al., 1999). Nevertheless, the distribution of surface charges in profilin-I and profilin-II is significantly different and this perhaps participates in the ligandbinding specificity (Figure 2). Eventually, identifying the structural features of different profilin complexes will be helpful to understand the basis of specificity.

Finally, the profilin- ligands binding should be a dynamic process and the mechanisms of ligand release under physiological conditions have to be determined. For example, actin can be released from profilactin complex via PtdIns(4,5)P2, and an analogous mechanism might be used for ligand binding regulation. For instance, PtdIns(4,5)P2 can regulate the interaction between dynamin 1 and profilin-II, but not the Mena–profilin or VASP–profilin complexes. Regulation of Mena, VASP, and other ligands binding might be achieved in different ways such as profilin or ligand phosphorylation (Witke, 2004).

#### **2.4 Role of profilin in signal transduction**

Profilins bind to several ligands, and a lot of these ligands are part of various complexes or interact with each other as well (Figure 3). This results in an intimate crosstalk among these complexes that can substitute and distribute components and, thus, could assimilate signals from other signaling pathways such as small-GTPase and phosphoinositide pathways. In these signaling platforms profilins appear to be a common denominator (Witke, 2004). Figure 6 is a schematic representation demonstrating various interactions between profilin, the microfilament system and different signaling pathways.

Profilins are linked to the phosphatidylinositol cycle and in turns to the receptor tyrosine kinase pathway through their binding to PtdIns 4,5-P2. Profilin-bound PtdIns 4,5-P2 is

release. Initially, the structural requirements for the profilin-ligand binding are not completely understood. In spite of binding of profilin to an extremely diverse group of ligands either directly or as part of a larger complex, the binding sites on both profilin and ligands appear to be well conserved. The majority of ligands are believed to interact with the PLP domain of profilin that contains the N- and C-terminal helices. The only exception, so far, to this model is gephyrin, which appears to bind to a special profilin domain (Giesemann et al., 2003). All profilin ligands are characterized by the presence of stretched or nearly stretched proline-rich domains that are required for profilin binding. Still, a contiguous prolines stretch is insufficient. Depending on the data obtained from *in vitro* studies using synthetic PLP peptides of different length high-affinity binding requires a decamer as a minimum, (Perelroizen et al., 1994) however this cannot be extended to cover proteins or to be used for recognizing or evaluating the ability of profilin to bind to a ligand. A lot of profilin ligands contain in their proline-rich domains proline repeats of no more than three or four successive prolines. Further amino acids, mostly glycines, appear to be capable of replacing proline, and an efficient profilin-binding domain appears to include numerous repeats that have the consensus sequence ZPPX (where Z=P, G or A; and X= any

The second important factor in regulating the binding of profilins to their ligands is the binding specificity of ligands to the different profilins. Previous reports showed that the interaction of ligands with profilin-I and profilin-II occurs in a highly specific manner (Witke et al., 1998) and it looks likely that it is not only the PLP-binding domains but also other complex binding parameters have to be considered. Comparative studies on the structures of mammalian profilin-I and profilin-II indicated that they are approximately superimposable (Nodelman et al., 1999). Nevertheless, the distribution of surface charges in profilin-I and profilin-II is significantly different and this perhaps participates in the ligandbinding specificity (Figure 2). Eventually, identifying the structural features of different

Finally, the profilin- ligands binding should be a dynamic process and the mechanisms of ligand release under physiological conditions have to be determined. For example, actin can be released from profilactin complex via PtdIns(4,5)P2, and an analogous mechanism might be used for ligand binding regulation. For instance, PtdIns(4,5)P2 can regulate the interaction between dynamin 1 and profilin-II, but not the Mena–profilin or VASP–profilin complexes. Regulation of Mena, VASP, and other ligands binding might be achieved in different ways

Profilins bind to several ligands, and a lot of these ligands are part of various complexes or interact with each other as well (Figure 3). This results in an intimate crosstalk among these complexes that can substitute and distribute components and, thus, could assimilate signals from other signaling pathways such as small-GTPase and phosphoinositide pathways. In these signaling platforms profilins appear to be a common denominator (Witke, 2004). Figure 6 is a schematic representation demonstrating various interactions between profilin,

Profilins are linked to the phosphatidylinositol cycle and in turns to the receptor tyrosine kinase pathway through their binding to PtdIns 4,5-P2. Profilin-bound PtdIns 4,5-P2 is

profilin complexes will be helpful to understand the basis of specificity.

such as profilin or ligand phosphorylation (Witke, 2004).

the microfilament system and different signaling pathways.

**2.4 Role of profilin in signal transduction** 

hydrophobic amino acid) (Witke et al., 1998).

resistant to hydrolysis by phospholipase Cγ1 (Goldschmidt-Clermont et al., 1990). However, this resistance can be overcome after activating phospholipase via receptor tyrosine kinasesdependant phosphorylation (Goldschmidt-Clermont et al., 1991). This activation process results in PtdIns 4,5-P2 hydrolysis with subsequent formation of other two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate. Additionally, profilin releases from the membrane, which might initiate fast, local actin polymerization. Conversely, activated phospholipase Cγ1 cannot hydrolyze other PI 3-kinase activity products such as PtdIns 3,4-P2 and PtdIns 3,4,5-P3 ,that bind to profilin with a higher affinity than PtdIns 4,5- P2. Consequently, it has been revealed that PtdIns 3,4-P2 and PtdIns 3,4,5-P3 may regulate phospholipase Cγ1-controlled turnover of PtdIns 4,5-P2 (Lu et al., 1996).

In addition, the profilin ligands of the formin-related proteins such as p140mDia connect the GTPase-related signaling cascade, which is also coupled with the PtdIns 4,5-P2 signaling pathway to the microfilament system. The small GTPases of the Rho family are active members that are involved in regulating the cytoskeleton-based processes such as cell morphology, adhesion and cytokinesis (Tapon & Hall, 1997). Most likely, these forminrelated proteins are down-stream effectors of Rho in this cascade (Evangelista et al., 1997; Watanabe et al., 1997).

Also, the microfilament system is linked to the adenylyl cyclase-related pathway via substrates of the cAMP/cGMP-dependent protein kinases such as VASP/Mena family (Butt et al., 1994; Gertler et al., 1996) and the putative profilin ligand CAP, which is an adenylyl cyclase activator (Fedor-Chaiken et al., 1990; Field et al., 1990; Toda et al., 1985). This linking can be executed through either direct binding of CAP and VASP proteins to actin (Freeman et al., 1995; Gieselmann & Mann, 1992; Gottwald et al., 1996; Hubberstey et al., 1996; Reinhard et al., 1992) or recruiting profilin and profilin-actin complexes to areas of dynamic actin remodelling via the interaction of VASP proteins with cell contact proteins such as zyxin and vinculin (Brindle et al., 1996; Gertler et al., 1996; Reinhard et al., 1995, 1996).

Furthermore, annexin I could be involved in this crosstalk depending on previous reports that described the sensitivity of annexin I-profilin binding to PtdIns 4,5-P2 and actin (Alvarez-Martinez et al., 1996). On top of that the annexins activity is controlled by the free Ca2+ level, which is adjusted via PtdIns 4,5-P2 hydrolysis upon the action of the activated phospholipase Cγ 1 (Figure 6). In addition to annexin I, Ca2+ level will affect various Ca2+ actin-binding and –severing proteins which slice the actin filaments and create new plus ends to which profilin-actin complexes can be added (Schlüter et al., 1997) (Figure 6).

Interestingly, in mesangial cells extracellular profilin was shown to bind specifically to a putative receptor and stimulates AP-1, a key element in signal transduction that is involved in the regulation of the transcription of several genes and cell growth (Tamura et al, 2000).

With the current large number of profilins ligands the future challenge is to determine their role in this complicated signaling crosstalk. One possibility is that profilins may act as regulators for the composition of the complexes and facilitate entrance or exit of certain ligands. Additionally, they might act as direct regulators for the ligands activities. Identification of all profilins molecular interactions, their ligands, and recognizing the structure of these complexes will be helpful to understand the mechanisms by which profilins can control this diverse signaling complexes (Witke, 2004).

Fig. 6. The involvement of profilin (red) in different signaling routes. This schematic drawing depicts only the main connections established so far. Molecules and second messengers of the polyphosphoinositide signaling pathway are indicated in yellow, protein members of signaling routes are marked green, proline-cluster proteins identified as profilin ligands are marked purple, the actin cycle is seen in blue, Ca2+ in intracellular stores and Ca2+ regulated microfilament proteins are marked grey. For simplicity, the solid arrows indicate either direct interactions between components, as shown by biochemical assays, or point to pathways. Broken arrows indicate suspected or indirect interactions. Abbreviations: PI-3,4,5-P3: phosphatidylinositol 3,4,5-trisphosphate; PIP2: phosphatidylinositol 4,5 bisphosphate; RTK: receptor tyrosine kinase; DAG: diacylglycerol; PLCγ 1: phospholipase Cγ 1; cAMP/cGMP- PK: cAMP/cGMP dependent protein kinase; IP3: inositol 1,4,5 trisphosphate, adapted from Schlüter et al., (1997) with permission.
