Profilin II Is Alternatively Spliced, Resulting in Profilin Isoforms That Are Differentially Expressed and Have Distinct Biochemical Properties

Abstract

We deduced the structure of the mouse profilin II gene. It contains five exons that can generate four different transcripts by alternative splicing. Two transcripts encode different profilin II isoforms (designated IIa and IIb) that have similar affinities for actin but different affinities for polyphosphoinositides and proline-rich sequences. Profilins IIa and IIb are also present in humans, suggesting that all mammals have three profilin isoforms. Profilin I is the major form in all tissues, except in the brain, where profilin IIa is most abundant. Profilin IIb appears to be a minor form, and its expression is restricted to a limited number of tissues, indicating that the alternative splicing is tightly regulated. Western blotting and whole-mount in situ hybridization show that, in contrast to the expression of profilin I, the expression level of profilin IIa is developmentally regulated. In situ hybridization of adult brain sections reveals overlapping expression patterns of profilins I and IIa.Changes in both cell shape and motility in response to extracellular signals require mechanical forces from the actin cytoskeleton. The formations of lamellipodia, focal contacts, the contractile ring during cytokinesis, and neuronal growth cone motility depend on the controlled polymerization-depolymerization of actin filaments (7,40). The regulation of these processes is accomplished by many actin binding proteins that act at different points in time and space, depending on the extracellular signals (44).Profilin is a small, ubiquitous actin binding protein, thought to be a key regulatory of actin dynamics in living cells (8,41). In vitro experiments have shown that profilin acts as an actin monomer-sequestering protein when barbed ends of filaments are capped (e.g., by gelsolin). When the capping protein is removed, polymerization of actin can occur, and actin-profilin complexes can add to the fast-growing ends, thereby enhancing actin polymerization in the presence of thymosin ␤4 (22, 34).Profilin can bind other molecules including phosphatidylinositol 4,5-bisphosphate (PIP 2 ) (28) and proline-rich domains of several proteins like the vasodilator-stimulated phosphoprotein (VASP), Enabled (Ena), mammalian Ena (Mena), aczonin, the Wiskott-Aldrich syndrome protein (WASP), its neural homologue N-WASP, and mammalian Diaphanous (p140mDia) (1,2,13,38,45,46,48). In lower eukaryotes, many other proline-rich proteins have been identified as profilin ligands, such as formin-homology proteins (47). The actinrelated protein-2/3 complex (Arp2/3 complex), composed of seven different polypeptides, was originally identified as a profilin binding complex (29).Until 1993, only one profilin isoform was known to be present in mammalian cells. Honoré and coworkers (20) discovered a second profilin gene in a random cDNA cloning project. The open reading frame predicted a protein with a high similarity to profilin I (62.1% identity), and therefore the protein was designated profilin II. Northern blot analysis showed that the expression l...

On the origin and evolution of vertebrate and viral profilins

“…In mammals, the profilin II gene gives rise to two isoforms due to alternative splicing [6,8]. To reveal conserved profilin II gene structures, we searched the Ensembl entries for chicken, Xenopus and zebrafish.…”

“…separately from profilins I and II The gene structures of mouse profilin I and II were described previously [6,8,22]. Basically, the splice sites are conserved between mouse profilin I and II and thus these forms have homologous introns, with the exception that profilin II contains an additional exon encoding the C-terminus of splice variant IIb.…”

“…Yet the biochemical properties, with respect to polyphosphoinositide binding and poly-L L-proline binding, of these profilin isoforms are very different (see e.g. [8,17] and Table 2). Obviously these differences are due to different sequence features, but apparently these are not sufficiently discriminative in the calculation of evolutionary distances.…”

“…Next to the vertebrate founding member of this protein family: profilin I [5], various profilins were identified in several species from the eukaryotic kingdom and in viruses. More recently, paralogues have been characterized in bovine, human, mouse and rat: the two splice variants profilins IIa and IIb, profilin III and profilin IV [6][7][8][9][10][11]. Previous phylogenetic analysis separated the profilins largely into two groups: profilins from vertebrates and from invertebrates.…”

“…Such studies have pointed to the fact that sequence similarity between profilins from invertebrate and vertebrate species is low, yet there is a remarkable conservation of 3D-structure and function [13,14]. For instance, the biochemically and structurally well-characterized profilins I and II from Acanthamoeba and Mammalia adopt the same fold [13] and bind actin, polyphosphoinositides and poly-L L-proline sequences [15], albeit that, in given species, for some isoforms differences in affinities for these ligands exist [8,16,17]. Aligning the primary structures of Acanthamoeba and Mammalian profilins, however, yields a similarity score lower than 25% and is thus in the twilight zone of evolutionary relationship based on sequence comparison.…”