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Drosophila enabled/vasodilator-stimulated phosphoprotein homology 1 (EVH1) domains are 115 residue protein-protein interaction modules which provide essential links for their host proteins to various signal transduction pathways. Many EVH1-containing proteins are associated closely with actin-based structures and are involved in re-organization of the actin cytoskeleton. EVH1 domains are also present in proteins enriched in neuronal tissue, thus implicating them as potential mediators of synaptic plasticity, linking them to memory formation and learning. Like Src homology 3, WW and GYF domains and profilin, EVH1 domains recognize and bind specific proline-rich sequences (PRSs). The binding is of low affinity, but tightly regulated by the high specificity encoded into residues in the protein:peptide interface. In general, a small (3-6 residue) 'core' PRS in the target protein binds a 'recognition pocket' on the domain surface. Further affinity- and specificity-increasing interactions are then formed between additional domain epitopes and peptide 'core-flanking' residues. The three-dimensional structures of EVH1:peptide complexes now reveal, in great detail, some of the most important features of these interactions and allow us to better understand the origins of specificity, ligand orientation and sequence degeneracy of target peptides, in low affinity signalling complexes.

We propose a normalization of symbols and terms used to describe, accurately and succinctly, the detailed interactions between amino acid residues of pairs of interacting proteins at protein:protein (or protein:peptide) interfaces. Our aim is to unify several diverse descriptions currently in use in order to facilitate communication in the rapidly progressing field of signaling by protein domains. In order for the nomenclature to be convenient and widely used, we also suggest a parallel set of symbols restricted to the ASCII format allowing accurate parsing of the nomenclature to a computer-readable form. This proposal will be reviewed in the future and will therefore be open for the inclusion of new rules, modifications and changes.

We previously identified a 42 kDa Ins(1,3,4,5)P4 (InsP4) receptor protein (p42IP4) in brain membranes from several species. Here the cDNA sequence of p42IP4 was obtained by PCR using degenerate primers derived from peptide sequences of proteolytic fragments of the porcine protein and by subsequent screening of a pig brain cDNA library. The derived peptide sequence of 374 amino acids for porcine p42IP4 is 45 amino acids shorter at the C-terminus than centaurin-alpha from rat (84% homology) and has a calculated molecular mass of 43 kDa. From the InsP4 binding activity present in brain tissue homogenate about 25% is found in the cytosolic fraction and 75% associated with microsomes. Both activities are due to p42IP4 since (i) a peptide-specific antiserum recognizing specifically p42IP4 labels the InsP4 receptor protein in membranes and in the cytosol, (ii) the antiserum immunoprecipitates both the membrane protein and the cytosolic protein of 42 kDa, (iii) the InsP4 binding activity released by high salt or by alkaline extraction from membranes is identified immunologically as the 42 kDa protein, and (iv) the affinity for InsP4 and specificity for various inositolphosphates are similar for the membrane-associated and for the soluble p42IP4. The functional importance of p42IP4 is highlighted by the identical affinity for InsP4 and for phosphatidylinositol (3,4,5)P3 (Ki = 1.6 and 0.9 nM, respectively). Thus, the InsP4 receptor, apparently a peripheral membrane protein, which exists also as a cytosolic protein can transfer the signals mediated by InsP4 or by PtdInsP3 between membranes and cytosolic compartment.

Detailed studies of differences in distinct cGMP kinase isoforms are highly dependent on expression of large amounts of these enzyme isoforms that are not easily purified by conventional methods. Here cGMP-dependent protein kinases, the type I beta soluble form from human placenta, and the type II membrane-associated form from rat intestine, were each expressed in a baculovirus/Sf9 cell system and purified in milligram amounts by affinity chromatography. The expressed recombinant proteins displayed characteristics like those of their native counterparts. cGK I beta was expressed as a 76 kDa protein predominantly found in the cytosol fraction, whereas cGK II was expressed as an 86 kDa protein predominantly associated with the membrane fraction. The apparent Ka and Vmax of cGMP for activation of cGK I beta were 0.5 microM and 3.4 mumol/min/mg, and for cGK II were 0.04 microM and 1.8 mumol/min/mg.