Vitronectin is a multifunctional glycoprotein present in blood and in the extracellular matrix. It binds glycosaminoglycans, collagen, plasminogen and the urokinase-receptor, and also stabilizes the inhibitory conformation of plasminogen activation inhibitor-1. By its localization in the extracellular matrix and its binding to plasminogen activation inhibitor-1, vitronectin can potentially regulate the proteolytic degradation of this matrix. In addition, vitronectin binds to complement, to heparin and to thrombin-antithrombin III complexes, implicating its participation in the immune response and in the regulation of clot formation. The biological functions of vitronectin can be modulated by proteolytic enzymes, and by exo- and ecto-protein kinases present in blood. Vitronectin contains an RGD sequence, through which it binds to the integrin receptor alpha v beta 3, and is involved in the cell attachment, spreading and migration. Antibodies against alpha v beta 3 or synthetic peptides containing an RGD sequence are now being tested as therapeutic agents in the treatment of human cancers, bone diseases (e.g. osteoporosis) and in pathological disorders which involve angiogenesis.

The amino acid sequences of all known cGMP-binding phosphodiesterases (PDEs) contain internally homologous repeats (a and b) that are 80 -90 residues in length and are arranged in tandem within the putative cGMPbinding domains. In the bovine lung cGMP-binding, cGMP-specific PDE (cGB-PDE or PDE5A), these repeats span residues 228 -311 (a) and 410 -500 (b). An aspartic acid (residue 289 or 478) that is invariant in repeats a and b of all known cGMP-binding PDEs was changed to alanine by site-directed mutagenesis of cGB-PDE, and wild type (WT) and mutant cGB-PDEs were expressed in COS-7 cells. Purified bovine lung cGB-PDE (native) and WT cGB-PDE displayed identical cGMP-binding kinetics, with ϳ1.8 M cGMP required for half-maximal saturation. The D289A mutant showed decreased affinity for cGMP (K d > 10 M) and the D478A mutant showed increased affinity for cGMP (K d Ϸ 0.5 M) as compared to WT and native cGB-PDE. WT and native cGB-PDE displayed an identical curvilinear profile of cGMP dissociation which was consistent with the presence of distinct slowly dissociating (k off ‫؍‬ 0.26 h ؊1 ) and rapidly dissociating (k off ‫؍‬ 1.00 h ؊1 ) sites of cGMP binding. In contrast, the D289A mutant displayed a single k off ‫؍‬ 1.24 h ؊1 , which was similar to the calculated k off for the fast site of WT and native cGB-PDE, and the D478A mutant displayed a single k off ‫؍‬ 0.29 h ؊1, which was similar to that calculated for the slow site of WT and native cGB-PDE. These results were consistent with the loss of a slow cGMP-binding site in repeat a of the D289A mutant cGB-PDE, and the loss of a fast site in repeat b of the D478A mutant, suggesting that cGB-PDE possesses two distinct cGMP-binding sites located at repeats a and b, with the invariant aspartic acid being crucial for interaction with cGMP at each site. Cyclic nucleotide phosphodiesterases (PDEs)1 constitute a complex family of enzymes which catalyze the hydrolysis of 3Ј:5Ј-cyclic nucleotides to the corresponding nucleoside 5Ј-monophosphates. The multiple PDEs differ in their substrate specificities, sensitivities to inhibitors, modes of regulation, and tissue distributions. Most PDEs are chimeric multidomain proteins, possessing distinct catalytic and regulatory domains (1). A 250-amino acid segment of sequence, which is conserved among all mammalian PDEs and is located in the more carboxyl-terminal portions of the PDE molecules, contains the catalytic site of these enzymes (1-4). Domains of the PDEs which interact with allosteric/regulatory factors are thought to be located within the more amino-terminal regions (1, 5, 6).The cGMP-binding PDEs comprise a heterogeneous subgroup of PDEs, all of which exhibit allosteric cGMP-binding sites that are distinct from the sites of cyclic nucleotide hydrolysis. This group consists of at least three classes of PDEs: the cGMP-stimulated PDEs (cGS-PDEs, or PDE2s 2 ) (7), the photoreceptor PDEs (rod outer segment PDE (ROS-PDE; PDE6A/B) (8) and cone PDE (PDE6C) (9)), and the cGMP-binding, cGMPspecific PDE (cGB-PDE; PDE5A) (10). The s...

We report on a short host defense-like peptide that targets and arrests the growth of aggressive and hormone-resistant primary human prostate and breast tumors and prevents their experimental and spontaneous metastases, respectively, when systemically inoculated to immuodeficient mice. These effects are correlated with increased necrosis of the tumor cells and a significant decrease in the overall tumor microvessel density, as well as newly formed capillary tubes and prostate-specific antigen secretion (in prostate tumors). Growth inhibition of orthotopic tumors derived from stably transfected highly fluorescent human breast cancer cells and prevention of their naturally occurring metastases were visualized in real time by using noninvasive whole-body optical imaging. The exclusive selectivity of the peptide towards cancer derives from its specific binding to surface phosphatidylserine and the killing of the cancer cells via cytoplasmic membrane depolarization. These data indicate that membrane disruption can provide a therapeutic means of inhibiting tumor growth and preventing metastases of various cancers. (Cancer Res 2006; 66(10): 5371-8)

Using a bovine 61-kDa (PDE1A2) calmodulin-stimulated phosphodiesterase (CaM-PDE) cDNA and a bovine lung 59-kDa (PDE1A1) CaM-PDE cDNA reported here, we have identified two new regions within the primary structure of these two related isozymes that are important for regulation by Ca 2؉ /CaM. PDE1A1 is identical to the PDE1A2 isozyme except for the amino-terminal 18 residues. In agreement with earlier studies, the CaM concentration required for half-maximal activation (K CaM ) of recombinant PDE1A1 (0.3 nM) was Ϸ10-fold less than the K CaM for recombinant PDE1A2 (4 nM). A series of deletion mutations of the PDE1A2 cDNA removing nucleotide sequence encoding the first 46 -106 aminoterminal residues were constructed and expressed using the baculovirus system. Deletion of the amino acids encompassing a previously identified, putative CaMbinding domain (residues 4 -46) produced a polypeptide that was still activated 3-fold by CaM (K CaM Ϸ 3 nM). However, complete CaM-independent activation occurred when residues 4 -98 were deleted. To determine the location of the additional CaM-binding domain(s), the inhibitory potency of seven overlapping, synthetic peptides spanning amino acids 76 -149 of PDE1A2 was tested using the CaM-activated enzyme. One peptide spanning amino acids 114 -137 of PDE1A2 appeared to be the most potent inhibitor of CaM-stimulated activity. These results reveal the existence of a CaM-binding domain located approximately 90 residues carboxyl-terminal to the putative CaM-binding domains previously identified within the PDE1A1 and PDE1A2 isozymes. Moreover, a discrete segment important for holding these CaM-PDEs in a less active state at low Ca 2؉ concentrations is located between the two CaM-binding domains.Calmodulin-stimulated cyclic nucleotide phosphodiesterases (CaM-PDEs) 1 constitute a genetically diverse class of cAMP and cGMP hydrolyzing activities that are activated by calcium and calmodulin (1). Several different CaM-PDE activities have been identified (2). These CaM-PDE isozymes are distributed among discrete cell types in various tissues and have varying substrate specificity, specific activities, and activation constants (K CaM ) for CaM (2, 3). In cells that express CaM-PDEs, hormones that increase cytosolic Ca 2ϩ would be predicted to activate this isozyme, thereby decreasing cyclic nucleotide accumulation in response to hormones that stimulate cAMP or cGMP synthesis.In addition to Ca 2ϩ /CaM, certain CaM-PDEs may be subject to a secondary form of regulation via phosphorylation. In vitro, the 59-kDa (PDE1A1) and 61-kDa (PDE1A2) CaM-PDEs 2 are phosphorylated by cAMP-dependent protein kinase (4, 5), while the autophosphorylated form of CaM-dependent protein kinase II catalyzes the phosphorylation of the 63-kDa (PDE1B1) CaM-PDE (6). Phosphorylation increases the K CaM , effectively rendering these isozymes less sensitive to activation by Ca 2ϩ (7,8). In vivo, CaM-PDE phosphorylation would likely result in potentiation of cAMP or cGMP accumulation in response to hormonal stimuli involving coin...

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