Transforming growth factor-betas (TGF-beta) are secreted as latent complexes consisting of the TGF-beta dimer, the TGF-beta propeptide dimer, and the latent TGF-beta binding protein (LTBP). Although the bonds between TGF-beta and its propeptide are cleaved intracellulary, the propeptide associates with TGF-beta by electrostatic interactions, thereby conferring latency to the complex. We reported that a specific sequence of LTBP-1 is required for latent TGF-beta activation by the integrin alphavbeta6. Here we describe a 24 amino acid sequence from the hinge domain required for activation. The LTBP-1 polypeptide rL1N, which includes the hinge, associates with fibronectin in binding assays. We present evidence that fibronectin null cells minimally activate latent TGF-beta and poorly incorporate the active hinge sequence into their matrix. In addition, cells missing the fibronectin receptor alpha5beta1 exhibit defective activation of latent TGF-beta by alphavbeta6 and decreased matrix incorporation. The results indicate specificity for integrin-mediated latent TGF-beta activation that include unique sequences in LTBP-1 and an appropriate matrix molecule.

Transforming growth factor-␤ (TGF-␤) activity is controlled at many levels including the conversion of the latent secreted form to its active state. TGF-␤ is often released as part of an inactive tripartite complex consisting of TGF-␤, the TGF-␤ propeptide, and a molecule of latent TGF-␤ binding protein (LTBP). The interaction of TGF-␤ and its cleaved propeptide renders the growth factor latent, and the liberation of TGF-␤ from this state is crucial for signaling. To examine the contribution of LTBP to TGF-␤ function, we generated mice in which the cysteines that link the propeptide to LTBP were mutated to serines, thereby blocking covalent association. Tgfb1 C33S/C33S mice had multiorgan inflammation, lack of skin Langerhans cells (LC), and a shortened lifespan, consistent with decreased TGF-␤1 levels. However, the inflammatory response and decreased lifespan were not as severe as observed with Tgfb1 ؊/؊ animals. Tgfb1 C33S/C33S mice exhibited decreased levels of active TGF-␤1, decreased TGF-␤ signaling, and tumors of the stomach, rectum, and anus. These data suggest that the association of LTBP with the latent TGF-␤ complex is important for proper TGF-␤1 function and that Tgfb1 C33S/C33S mice are hypomorphs for active TGF-␤1. Moreover, although mechanisms exist to activate latent TGF-␤1 in the absence of LTBP, these mechanisms are not as efficient as those that use the latent complex containing LTBP.

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The molecular mechanism of the presynaptic neurotoxicity of snake venom phospholipases A2 (PLA2s) is not yet fully elucidated. Recently, new high‐affinity binding proteins for PLA2 toxins have been discovered, including the important intracellular Ca2+ sensor, calmodulin (CaM). In the present study, the mode of interaction of group IIA PLA2s with the Ca2+‐bound form of CaM was investigated by mutational analysis of ammodytoxin A (AtxA) from the long‐nosed viper (Vipera ammodytes ammodytes). Several residues in the C‐terminal part of AtxA were found to be important in this interaction, particularly those in the region 115–119. In support of this finding, introduction of Y115, I116, R118 and N119, present in AtxA, into a weakly neurotoxic PLA2 from Russell's viper (Daboia russellii russellii) increased by sevenfold its binding affinity for CaM. Furthermore, two out of four peptides deduced from different regions of AtxA were able to compete with the toxin in binding to CaM. The nonapeptide showing the strongest inhibition was that comprising the AtxA region 115–119. This stretch contributes to a distinct hydrophobic patch within the region 107–125 in the C‐terminal part of the molecule. This lacks any substantial helical structure and is surrounded by several basic residues, which may form a novel binding motif for CaM on the molecular surface of the PLA2 toxin.