Importance of the C-terminus of the human 5-HT3A receptor subunit.

Amongst the family members of Cys-loop LGICs, the atypical ability of the 5-HT3A subunit to form functional homomeric receptors allowed a direct investigation of the role of the C-terminus. Deletion of the three C-terminal amino acids (DeltaGln453-DeltaTyr454-DeltaAla455) from the h5-HT3A subunit prevented formation of a specific radioligand binding site as well as expression within the cell membrane. Removal of merely the C-terminal residue (DeltaAla455) reduced specific radioligand binding (to 4+/-1% relative to the wild-type; cells grown at 37 degrees C) and also cell membrane expression; these reductions were less evident when the DeltaAla455 expressing cells were grown at 27 degrees C (specific radioligand binding levels 27+/-5% relative to wild-type also grown at 27 degrees C). Mutation of the h5-HT3A C-terminal amino acid, alanine, for either glycine (Ala455Gly), valine (Ala455Val) or leucine (Ala455Leu) reduced specific radioligand binding levels by 24+/-23%, 32+/-12% and 88+/-1%, respectively; the latter mutant also displaying reduced membrane expression. In contrast, mutation to alanine of the two amino acids preceding the C-terminal alanine (Gln453Ala and Tyr454Ala) had no detrimental effects on specific radioligand binding or cell membrane expression levels. The present study demonstrates an important role for the C-terminus in the formation of the functional h5-HT3A receptor. The partial restoration of 5-HT3 receptor binding and cell membrane expression when cells expressing C-terminal mutant 5-HT3A subunits were grown at a lower temperature (27 degrees C) suggests that the C-terminus stabilises the 5-HT3 receptor allowing subunit folding and subsequent maturation.

The regulatory effects of tropomyosin and troponin-I on the interaction of myosin loop regions with F-actin.

The N terminus of skeletal myosin light chain 1 and the cardiomyopathy loop of human cardiac myosin have been shown previously to bind to actin in the presence and absence of tropomyosin (Patchell, V. B., Gallon, C. E., Hodgkin, M. A., Fattoum, A., Perry, S. V., and Levine, B. A. (2002) Eur. J. Biochem. 269, 5088-5100). We have extended this work and have shown that segments corresponding to other regions of human cardiac beta-myosin, presumed to be sites of interaction with F-actin (residues 554-584, 622-646, and 633-660), likewise bind independently to actin under similar conditions. The binding to F-actin of a peptide spanning the minimal inhibitory segment of human cardiac troponin I (residues 134-147) resulted in the dissociation from F-actin of all the myosin peptides bound to it either individually or in combination. Troponin C neutralized the effect of the inhibitory peptide on the binding of the myosin peptides to F-actin. We conclude that the binding of the inhibitory region of troponin I to actin, which occurs during relaxation in muscle when the calcium concentration is low, imposes conformational changes that are propagated to different locations on the surface of actin. We suggest that the role of tropomyosin is to facilitate the transmission of structural changes along the F-actin filament so that the monomers within a structural unit are able to interact with myosin.

The inhibitory region of troponin-I alters the ability of F-actin to interact with different segments of myosin.

Peptides corresponding to the N-terminus of skeletal myosin light chain 1 (rsMLC1 1-37) and the short loop of human cardiac beta-myosin (hcM398-414) have been shown to interact with skeletal F-actin by NMR and fluorescence measurements. Skeletal tropomyosin strengthens the binding of the myosin peptides to actin but does not interact with the peptides. The binding of peptides corresponding to the inhibitory region of cardiac troponin I (e.g. hcTnI128-153) to F-actin to form a 1 : 1 molar complex is also strengthened in the presence of tropomyosin. In the presence of inhibitory peptide at relatively lower concentrations the myosin peptides and a troponin I peptide C-terminal to the inhibitory region, rcTnI161-181, all dissociate from F-actin. Structural and fluorescence evidence indicate that the troponin I inhibitory region and the myosin peptides do not bind in an identical manner to F-actin. It is concluded that the binding of the inhibitory region of troponin I to F-actin produces a conformational change in the actin monomer with the result that interaction at different locations of F-actin is impeded. These observations are interpreted to indicate that a major conformational change occurs in actin on binding to troponin I that is fundamental to the regulatory process in muscle. The data are discussed in the context of tropomyosin's ability to stabilize the actin filament and facilitate the transmission of the conformational change to actin monomers not in direct contact with troponin I.

Phosphorylation of the minimal inhibitory region at the C-terminus of caldesmon alters its structural and actin binding properties.

Caldesmon is an inhibitory protein believed to be involved in the regulation of thin filament activity in smooth muscles and is a major cytoplasmic substrate for MAP kinase. NMR spectroscopy shows that the actin binding properties of the minimal inhibitory region of caldesmon, residues 750-779, alter upon MAP kinase phosphorylation of Ser-759, a residue not involved in actin binding. This phosphorylation leads to markedly diminished actin affinity as a result of the loss of interaction at one of the two sites that bind to F-actin. The structural basis for the altered interaction is identified from the observation that phosphorylation destabilises a turn segment linking the two actin binding sites and thereby results in the randomisation of their relative disposition. This modulatory influence of Ser-759 phosphorylation is not merely a function of the bulkiness of the covalent modification since the stability of the turn region is observed to be sensitive to the ionisation state of the phosphate group. The data are discussed in the context of the inhibitory association of the C-terminal domain of caldesmon with F-actin.