Profilin (mw 12 000-15 000) is an abundant actin monomer-binding protein in eukaryotic cells (see Actin-Binding Proteins) ((1); review). Particularly high concentrations of profilin are found in lymphoid cells and brain cells. In lymphoid tissues the concentration of unpolymerized actin is about 100 mM and in platelets it may be as high as 200 |iM. In both cases about 50% of the unpolymerized actin can be accounted for by profilin:b-actin and profilin:g-actin isoforms. The remaining unpolymerized actin appears to be sequestered by b-thymosins (see below). In tissue cultured cells, profilin is concentrated in regions of high motile activity (2), where it is thought to bring actin monomers to growing ( +)-ends of filaments exposed through transmembrane signalling events. It binds to a number of cell cortex-associated proteins participating in the control of actin filament formation, eg the VASP family of proteins (3), the WASP-binding protein WIP (4), the formins (5-7) and the Arp2 protein (8).
Sacharomyces cerevisiae has one essential profilin gene (9), Dictyostelium discoideum has two (10), and plants contain multiple isoforms of profilin (10). Acanthamoeba castellani also contains two isoforms of profilin (11), as do mammalian cells (12).
Genetic studies suggest that profilin plays a key role in the establishment of actin-dependent cell polarity in Saccharomyces cerevisiae, in the formation of the fruiting body in Dichtyostelium discoideum, and during early development in Drosophila (10, 13). In transgenic mice, homozygous profilin "knock-outs" are lethal emphasizing the physiological importance of profilin in mammalian cells (14).
The structure and ligand-binding by different isoforms of profilins have been elucidated by mutagenesis (15), crystallography (16-19) and NMR (20). All isoforms of profilin have similar folds even though the amino acid sequence is highly divergent. In Arabidopsis profilin, the residues involved in the actin interaction have diverged, but the general character of involved residues are conserved. In the profilin:b-actin crystal, profilin makes two contacts with actin. The largest contact involves 2260 A2 of buried surface area, and is the contact between profilin and actin in solution. It engages subdomains 1 and 3 of actin at the (+)-end of the actin monomer (16). This leaves the (-)-end of the monomer free to bind to the fast growing ( +)-end of an actin filament in the polymerization process. The less extensive profilin:actin interaction site (1187 A 2 b.s.a.) seen in the crystal is formed by the N-terminal helix of profilin binding to subdomain 4 of a different actin monomer (16). So far, this contact has not been observed in solution.
In vitro, profilin inhibits the polymerization of actin with varying efficiency depending on the ionic conditions, and on whether the homologous nonmuscle actin or heterologous muscle a-actin is used (21-23). In the presence of physiological concentrations of Mg ions, profilin is a relatively poor actin sequesterer. Apparently, the conformation of the (-)-end is not disturbed by profilin, and the profilin:actin complex can add onto the (+)-end of filaments. Thus both actin and the profilin:actin complex can support growth of actin filaments at the fast growing ( +)-end (22, 24). The characteristics of the association/dissociation of actin monomers at the (-)-end of actin filaments are such that profilin effectively prevents the addition of actin at that end.
Beta thymosins, which inhibit growth at both ends and occur at high concentrations in cells, are thought to be the primary actin-sequestering proteins, and profilin, with its higher affinity for actin, serves to deliver actin to the barbed end (24). In the presence of proteins that cap the (+)-end of actin filaments, profilin is an efficient actin sequestering protein, since it effectively prevents polymerization at the (-)-end under all conditions (25, 26). This implies that the control of actin polymerization in cells may depend primarily on regulating the association/dissociation of proteins that cap the (+)-end of preexisting filaments, or by regulating the nucleation of new filaments.
Profilin increases the rate of nucleotide exchange on actin more than 1 000-fold (27, 28), and stabilizes the actin in the nucleotide-free state. Since actin with ATP bound polymerizes faster than ADP-actin, profilin might promote repolymerization of actin.ADP monomers coming off of filaments during depolymerization, by rapidly converting them to ATP-actin monomers.
In a search for proline hydroxylase, Tanaka and Shibata found that affinity columns of poly(L-proline) (PLP), in addition to the hydroxylase, also bound profilin and actin. This allowed the development of a simple procedure to obtain a mixture of profilin and the two isoforms of profilin:nonmuscle actin (29, 30), from which the profilin:b-actin and profilin:g-actin isoforms can be isolated (31). The poly(L-proline) binding site on profilin has been identified. It is comprised of highly invariant hydrophobic and aromatic amino acids involving the N- and the C-terminal helices of profilin (15). Mutagenesis, modelling and direct structure determination has shown a bonding pattern in the profilin:polyproline interaction that is analogous to that of SH3 domains in signal transduction proteins (15, 18, 19). The VASP family of proteins, the WIP protein, and the formins all contain proline rich sequences with capacity to bind profilin. Profilin has also been found to bind to Arp2/Arp3 complex which is a multiprotein complex implicated in the mechanism of actin polymerization (see below).
In vitro, profilin alone or in complex with nonmuscle actins binds the phospholipid PtdIns 4.5-bisphosphate in micellar form, as well as in vesicles together with other phospholipids. Profilin:b/g-actin complexes also bind to PtdIns 4,5-bisphosphate in vitro, something which results in dissociation of the complexs with release of polymerization-competent actin. This would seem to indicate that the appearance of PtdIns 4,5-bisphosphate in the plasma membrane could recruit profilin:actin for polymerization in the advancing cell edge. However, that this is an oversimplification is indicated by the observations that the hydrolysis of PtdIns 4,5-bisphosphate by phospholipase Cg-1 is inhibited when profilin is associated with the PtdIns 4,5-bisphosphate substrate, and that phosphorylation of the enzyme with an activated tyrosine kinase receptor overrides this inhibition. This points at profilin also playing a role in the control of the metabolism of the polyphosphoinositides. Furthermore, profilin binds to the regulatory subunit of PtdIns 4,5-bisphosphate kinase and stimulates the formation of PtdIns 3,4,5-trisphosphate ((1)-for refs).
These observations point at profilin being an important factor also in the control of the release of inositol trisphosphate and thus of the generation of Ca pulses in the cell. The primary targets for 2 +
Ca regulation in cells is the actomyosin system with global changes in structure and activity as the result. It is now known that many of the microfilament-associated proteins interact with components formed in the phosphatidylinositol-cycle as a result of receptor-mediated activation of the cell, and that these interactions modulate the activity of these proteins vis-a-vis actin. Although the exact physiological roles of these interactions remain to be elucidated, it suggests that the phosphatidylinositol-cycle is directly involved in controlling the microfilament-based motility cycle.
