One of the primary points of regulation of transforming growth factor-␤ (TGF-␤) activity is control of its conversion from the latent precursor to the biologically active form. We have identified thrombospondin-1 as a major physiological regulator of latent TGF-␤ activation. Activation is dependent on the interaction of a specific sequence in thrombospondin-1 (K 412 RFK415 ) with the latent TGF-␤ complex. Platelet thrombospondin-1 has TGF-␤ activity and immunoreactive mature TGF-␤ associated with it. We now report that the latency-associated peptide (LAP) of the latent TGF-␤ complex also interacts with thrombospondin-1 as part of a biologically active complex. Thrombospondin⅐LAP complex formation involves the activation sequence of thrombospondin-1 (KRFK) and a sequence (LSKL) near the amino terminus of LAP that is conserved in TGF-␤ 1-5 . The interactions of LAP with thrombospondin-1 through the LSKL and KRFK sequences are important for thrombospondin-mediated activation of latent TGF-␤ since LSKL peptides can competitively inhibit latent TGF-␤ activation by thrombospondin or KRFKcontaining peptides. In addition, the association of LAP with thrombospondin-1 may function to prevent the reformation of an inactive LAP⅐TGF-␤ complex since thrombospondin-bound LAP no longer confers latency on active TGF-␤. The mechanism of TGF-␤ activation by thrombospondin-1 appears to be conserved among TGF-␤ isoforms as latent TGF-␤ 2 can also be activated by thrombospondin-1 or KRFK peptides in a manner that is sensitive to inhibition by LSKL peptides.

The cystic fibrosis gene encodes a chloride channel, CFTR (cystic fibrosis transmembrane conductance regulator), that regulates salt and water transport across epithelial tissues. Phosphorylation of the cytoplasmic regulatory (R) domain by protein kinase A activates CFTR by an unknown mechanism. The amino-terminal cytoplasmic tail of CFTR was found to control protein kinase A-dependent channel gating through a physical interaction with the R domain. This regulatory activity mapped to a cluster of acidic residues in the NH(2)-terminal tail; mutating these residues proportionately inhibited R domain binding and CFTR channel function. CFTR activity appears to be governed by an interdomain interaction involving the amino-terminal tail, which is a potential target for physiologic and pharmacologic modulators of this ion channel.

Calcium activation of fast striated muscle results from an opening of the regulatory N-terminal domain of fast skeletal troponin C (fsTnC), and a substantial exposure of a hydrophobic patch, essential for Ca 2؉ -dependent interaction with fast skeletal troponin I (fsTnI). This interaction is obligatory to relieve the inhibition of strong, force-generating actin-myosin interactions. We have determined intersite distances in the N-terminal domain of cardiac TnC (cTnC) by fluorescence resonance energy transfer measurements and found negligible increases in these distances when the single regulatory site is saturated with Ca Contraction of striated muscle is regulated by a group of actin-binding proteins, the troponin-tropomyosin complex located on the actin filament (1). Troponin is a heterotrimer. The subunit TnC 1 binds Ca 2ϩ , TnI binds actin and inhibits actomyosin ATPase in relaxed muscle, and troponin T anchors the three-subunit complex to tropomyosin on the actin filament. These proteins form the thin filament. Strong force-generating interactions between myosin and actin are initiated by the binding of Ca 2ϩ to the regulatory sites located in the N-terminal regulatory domain of TnC. The binding of activator Ca 2ϩ to TnC weakens or breaks the interaction between TnI and actin, thus releasing the inhibition of actomyosin ATPase and initiating force generation.The crystal structure of TnC from avian fast skeletal muscle TnC shows a dumbbell-shaped molecule with the N-and Cterminal segments folded into two globular domains connected by a long ␣-helical central helix (2, 3). Each domain has two Ca 2ϩ -binding EF-hand (helix-loop-helix) motifs. The five helices in the N-domain are labeled the N-helix and helices A-D, starting from the N terminus (Fig. 1). The four helices in the C-domain are labeled helices E-H, starting from the C-terminal end of the central helix. The N-domain of fast skeletal TnC has two Ca ). The two sites in the N-domain are the regulatory sites. Site I consists of the helix A-loop-helix B and site II the helix C-loop-helix D motif. Sites III and IV in the C-domain are believed to play a structural role and are occupied by Mg 2ϩ under physiological conditions in relaxed muscle. Site I in cardiac TnC is inactive in chelating Ca 2ϩ due to substitutions of two critical amino acids and an insertion in the binding loop; saturation of site II by Ca 2ϩ is sufficient to trigger contraction in cardiac muscle (4). The crystal structure of fsTnC contains two bound Ca 2ϩ ions in the C-domain and no bound cation in the N-domain. Based on the structure of the C-domain, an early computer model (5) (the HMJ model) suggests that Ca 2ϩ binding to the regulatory sites induces reorientations of the B and C helices relative to the A and D helices, thus exposing a hydrophobic patch located in the B helix (see Fig. 1). The exposed hydrophobic patch in this open conformation becomes available for the Ca 2ϩ -dependent interaction with TnI. This model also has been used to interpret functional and drug binding proper...

[reaction: see text] A novel strategy to generate thioester peptides compatible with Fmoc chemistry is presented. Peptide-C(alpha)oxy-(2-mercapto-1-carboxyamide)ethyl ester undergoes an O to S acyl shift during ligation and the newly formed thioester intermediate reacts with an N-terminal cysteine fragment generating a product with native amide bond at the ligation site.

Polypeptides possessing N-terminal cysteine or threonine can be easily purified using this 'covalent capture' approach.