PDZ domains are protein-protein interaction modules that typically bind to short peptide sequences at the carboxyl terminus of target proteins. Proteins containing multiple PDZ domains often bind to different transmembrane and intracellular proteins, playing a central role as organizers of multimeric complexes. To characterize the rules underlying the binding specificity of different PDZ domains, we have assembled a novel repertoire of random peptides that are displayed at high density at the carboxyl terminus of the capsid D protein of bacteriophage . We have exploited this combinatorial library to determine the peptide binding preference of the seven PDZ domains of human INADL, a multi-PDZ protein that is homologous to the INAD protein of Drosophila melanogaster. This approach has permitted the determination of the consensus ligand for each PDZ domain and the assignment to class I, class II, and to a new specificity class, class IV, characterized by the presence of an acidic residue at the carboxyl-terminal position. Homology modeling and site-directed mutagenesis experiments confirmed the involvement of specific residues at contact positions in determining the domain binding preference. However, these experiments failed to reveal simple rules that would permit the association of the chemical characteristics of any given residue in the peptide binding pocket to the preference for specific amino acid sequences in the ligand peptide. Rather, they suggested that to infer the binding preference of any PDZ domain, it is necessary to simultaneously take into account all contact positions by using computational procedures. For this purpose we extended the SPOT algorithm, originally developed for SH3 domains, to evaluate the probability that any peptide would bind to any given PDZ domain.A large number of interactions in the cell are mediated by families of protein binding modules that are found repeatedly and in different combinations in several proteins. Typically these modules mediate protein-protein interactions through recognition of short peptides in the target protein (1). Several approaches, based upon the screening of repertoires of combinatorial peptides, have been developed to investigate the recognition specificity of these domain families. Phage display of small peptides of random sequence has been successfully used for the characterization of binding domains such as SH2, SH3, WW, EH, etc. (reviewed in Ref. 2). PDZ domains (identified as conserved elements in postsynaptic density protein PSD-95, Disc-large tumor suppressor Dlg, Zonula occludens protein ZO-1) differ from the remaining domains since they bind to specific carboxyl-terminal sequences of target proteins and/or dimerize with other PDZ domains (reviewed in Ref. 3). This peculiarity has limited the possibility of using "classical" peptide repertoires displayed by fusion to M13 coat proteins, since these display systems present random peptides by fusing them to the amino terminus of pIII or pVIII coat proteins. As a consequence, PDZ specificity ha...

Synapsin I is a synaptic vesicle-associated phosphoprotein that has been implicated in the formation of presynaptic specializations and in the regulation of neurotransmitter release. The nonreceptor tyrosine kinase c-Src is enriched on synaptic vesicles, where it accounts for most of the vesicle-associated tyrosine kinase activity. Using overlay, affinity chromatography, and coprecipitation assays, we have now shown that synapsin I is the major binding protein for the Src homology 3 (SH3) domain of c-Src in highly purified synaptic vesicle preparations. The interaction was mediated by the proline-rich domain D of synapsin I and was not significantly affected by stoichiometric phosphorylation of synapsin I at any of the known regulatory sites. The interaction of purified c-Src and synapsin I resulted in a severalfold stimulation of tyrosine kinase activity and was antagonized by the purified c-Src-SH3 domain. Depletion of synapsin I from purified synaptic vesicles resulted in a decrease of endogenous tyrosine kinase activity. Portions of the total cellular pools of synapsin I and Src were coprecipitated from detergent extracts of rat brain synaptosomal fractions using antibodies to either protein species. The interaction between synapsin I and c-Src, as well as the synapsin I-induced stimulation of tyrosine kinase activity, may be physiologically important in signal transduction and in the modulation of the function of axon terminals, both during synaptogenesis and at mature synapses.Synapsin I is the major synaptic vesicle protein that binds to the Src homology 3 (SH3) domains of the adapter protein Grb2 in vitro (1). This interaction is mediated by domain D of synapsin I, a 23-kDa proline-rich, strongly basic domain located in the COOH-terminal portion of synapsin I (2). It seemed possible that domain D or other proline-rich regions in synapsin I might interact with other SH3 domain-containing proteins within the nerve terminal and that these interactions might have a physiological role in presynaptic function. One such candidate, the SH3 domain-containing nonreceptor tyrosine kinase c-Src, is expressed at high levels in postmitotic neurons and is enriched on synaptic vesicles, where it accounts for most of the vesicle-associated tyrosine kinase activity (3-6). Using purified components in vitro, we now report that a domain Dmediated interaction of synapsin I with the SH3 domain of c-Src results in a 5-fold activation of the catalytic activity of c-Src. We also present studies employing a variety of biochemical techniques that serve to further characterize the specificity, subcellular location, regulation, and potential functional consequences of the c-Src͞synapsin I interaction.

The sexual and asexual phases of reproductive cycles of two sponges, Tethya citrina and T. auranrium, living sympatrically in a Mediterranean coastal lagoon (Stagnone di Marsala, NW Sicily), were studied from samples collected over an 18-mo period. Both species are oviparous and gonochoric. They have a summer, partially overlapping, period of oocyte production, although T. citrina appear to mature earlier.No males were found, possibly due to the very short period of spermatogenesis. Both species produce asexual buds during the autumn/winter months. However, they seem to follow different reproductive strategies, with T. citrina showing a significantly lower production of buds than T. aurantium; by contrast, egg production is significantly lower in the latter species. The difference in the reproductive resource allocation is consistent with data reported in the literature on the anatomy features, genetic population structure and ecological distribution.

Classi¢cation of protein interaction domains on the basis of the chemical characteristics of binding pocket residues is a di¤cult task, because multiple contact positions are usually involved in the recognition speci¢city mechanism. On the other hand, target peptides may be classi¢ed according to the (few) speci¢c residues that constitute the binding motif, through analysis of molecular repertoires (libraries of synthetic peptides; phage display; two hybrid, etc.) that allow identifying collections of di¡erent ligands.We have recently pointed out that, in order to characterize PDZ domains and to infer their binding speci¢city, it is necessary to exploit computational procedures, which simultaneously take into account all the contact positions of the domain binding pocket and the corresponding residues of the ligand [1]. PDZ domains are protein interaction modules that recognize and bind the C-terminal four residues of their target. The solution of X-ray crystallographic structure of PDZ domains complexed with their peptide ligands reveals at least 23 contact positions, whose interacting atoms are at a distance shorter than the sum of the van der Waals radii (r) +3 A î .Some of these positions contain residues that are highly conserved in the PDZ domain family: for example thè GLGF' loop and a positively charged residue that accommodate the terminal carboxylate group. The majority of the PDZ domains (identi¢ed in the proteome up to now) recognize ligands of class I (Table 1). These PDZ domains are provided with a hydrophilic pocket, where residues of the LB strand and of the KB helix (in particular a histidine, highly conserved at position KB1) are involved in contacting the peptide ligand.Most of the remaining PDZ domains recognize a varied class of ligand peptides, characterized by aromatic or hydrophobic residues at position P 32 . Even residues mimicking part of a hydrophobic moiety at position P 32 (such as the arginine at P 32 of the peptide ligand in the crystallized structure of hCASK) can be accommodated in the large hydrophobic pocket that characterizes PDZ domains binding to class II peptides [2]. Main determinants of the binding, as derived from the contacts in the crystal structure, are residues at LB5, KB1 and KB3 positions.Bezprozvanny and Maximov have recently proposed a classi¢cation of the PDZ domains listed in the SMART Website, based upon the type of residues present in only two contact positions^LB5 and KB1^of the binding pocket [3]. By grouping the couples of residues on the basis of their polarity and/or bulkiness, they de¢ned 25 groups and correlated them to experimentally determined ligands. Unfortunately, ligand sequences are available only for nine out of 25 groups and, while the ¢rst group (G,H) is enforced by the presence of 68 PDZ domains (those binding to class I motifs), the others are less clearly determined. Two of them (G,n) and (a,p) do not correspond to known PDZ