The Cell Adhesion Activity of the Joining Peptide of Proopiomelanocortin.

Proopiomelanocortin (POMC) is a precursor protein of several peptide hormones, such as ACTH and ß-endorphin. Almost all of the peptide hormones in POMC have been drastically investigated in terms of their biological activities. However, the biological activity of the joining peptide region (JP) in POMC is unknown. Therefore, to explore the biological activity of JP, sequence analyses of mammalian POMC were performed. We found an -Arg-Gly-Asp- (RGD) motif in several mammalian species, such as porcine, suggesting that JP has cell adhesion activity. To validate this hypothesis, the cell adhesion activities of the synthetic porcine JP peptides were examined using 293T cells. Cell adhesions were observed in a concentration-dependent manner of the JP peptides. In addition, the JP peptide competitively inhibited cell adhesion to the POMC-coated plates. Moreover, the cell adhesion activity of the joining peptide was inhibited by the addition of EDTA, indicating that the JP peptide mediates the cell adhesion activity via a receptor protein, integrin. Interestingly, a human JP peptide, which possesses an -Arg-Ser-Asp- (RSD) sequence in place of the RGD sequence, exhibited a higher ability in the cell adhesion activity than that of the porcine JP peptide, suggesting that the cell adhesion activity of the joining peptide is developed during the molecular evolution of POMC. In conclusion, our results reveal that the joining peptide in POMC plays an important role during cell adhesion and provide useful information related to signal transduction of nerve peptide hormones derived from POMC.

Ligand-dependent responses of the silkworm prothoracicotropic hormone receptor, Torso, are maintained by unusual intermolecular disulfide bridges in the transmembrane region.

The insect membrane-protein, Torso, is a member of the receptor-tyrosine-kinase family, and is activated by its ligand, prothoracicotropic hormone (PTTH). Although PTTH is one of the most important regulators of insect development, the mechanism of Torso activation by the hormone has remained elusive. In this study, using heterologous expression in cultured Drosophila S2 cells, we detected ligand-independent dimerization of silkworm Torso, and found that the receptor molecules in the dimer were linked by intermolecular disulfide bridges. By examining the oligomerization states of several truncation and substitution mutants of Torso, atypical cysteine residues in the transmembrane region were identified as being responsible for the intermolecular linkage in the dimer. The replacement of all of the cysteines in the region with phenylalanines abolished the disulfide-bond-mediated dimerization; however, non-covalent dimerization of the mutant was detected using a cross-linking reagent, both with and without ligand stimulation. This non-covalent dimerization caused apparent receptor autophosphorylation independently of the ligand stimulation, but did not promote the ERK phosphorylation in the downstream signaling pathway. The unique Torso structure with the intermolecular disulfide bridges in the transmembrane region is necessary to maintain the ligand-dependent receptor functions of autophosphorylation and downstream activation.

Effects of positively charged redox molecules on disulfide-coupled protein folding.

In vitro folding of disulfide-containing proteins is generally regulated by redox molecules, such as glutathione. However, the role of the cross-disulfide-linked species formed between the redox molecule and the protein as a folding intermediate in the folding mechanism is poorly understood. In the present study, we investigated the effect of the charge on a redox molecule on disulfide-coupled protein folding. Several types of aliphatic thiol compounds including glutathione were examined for the folding of disulfide-containing-proteins, such as lysozyme and prouroguanylin. The results indicate that the positive charge and its dispersion play a critical role in accelerating disulfide-coupled protein folding.