Morikatsu Yoshida

Deficiency of triad junction and contraction in mutant skeletal muscle lacking junctophilin type 1

In skeletal muscle excitation–contraction (E–C) coupling, the depolarization signal is converted from the intracellular Ca2+ store into Ca2+ release by functional coupling between the cell surface voltage sensor and the Ca2+ release channel on the sarcoplasmic reticulum (SR). The signal conversion occurs in the junctional membrane complex known as the triad junction, where the invaginated plasma membrane called the transverse-tubule (T-tubule) is pinched from both sides by SR membranes. Previous studies have suggested that junctophilins (JPs) contribute to the formation of the junctional membrane complexes by spanning the intracellular store membrane and interacting with the plasma membrane (PM) in excitable cells. Of the three JP subtypes, both type 1 (JP-1) and type 2 (JP-2) are abundantly expressed in skeletal muscle. To examine the physiological role of JP-1 in skeletal muscle, we generated mutant mice lacking JP-1. The JP-1 knockout mice showed no milk suckling and died shortly after birth. Ultrastructural analysis demonstrated that triad junctions were reduced in number, and that the SR was often structurally abnormal in the skeletal muscles of the mutant mice. The mutant muscle developed less contractile force (evoked by low-frequency electrical stimuli) and showed abnormal sensitivities to extracellular Ca2+. Our results indicate that JP-1 contributes to the construction of triad junctions and that it is essential for the efficiency of signal conversion during E–C coupling in skeletal muscle.

Identification of the novel bioactive peptides dRYamide-1 and dRYamide-2, ligands for a neuropeptide Y-like receptor in Drosophila

A number of bioactive peptides are involved in regulating a wide range of animal behaviors, including food consumption. Vertebrate neuropeptide Y (NPY) is a potent stimulator of appetitive behavior. Recently, Drosophila neuropeptide F (dNPF) and short NPF (sNPF), the Drosophila homologs of the vertebrate NPY, were identified to characterize the functions of NPFs in the feeding behaviors of this insect. Dm-NPFR1 and NPFR76F are the receptors for dNPF and sNPF, respectively; both receptors are G protein-coupled receptors (GPCRs). Another GPCR (CG5811; NepYR) was indentified in Drosophila as a neuropeptide Y-like receptor. Here, we identified 2 ligands of CG5811, dRYamide-1 and dRYamide-2. Both peptides are derived from the same precursor (CG40733) and have no significant structural similarities to known bioactive peptides. The C-terminal sequence RYamide of dRYamides is identical to that of NPY family peptides; on the other hand, dNPF and sNPF have C-terminal RFamide. When administered to blowflies, dRYamide-1 suppressed feeding motivation. We propose that dRYamides are related to the NPY family in vertebrates, similar to dNPF and sNPF.

Differential Intracellular Signaling through PAC1 Isoforms as a Result of Alternative Splicing in the First Extracellular Domain and the Third Intracellular Loop

Pituitary adenylate cyclase-activating polypeptide (PACAP), a pleiotropic neuropeptide, performs a variety of physiological functions. The PACAP-specific receptor PAC1 has several variants that result mainly from alternative splicing in the mRNA regions encoding the first extracellular (EC1) domain and the third intracellular cytoplasmic (IC3) loop. The effects on downstream signaling produced by combinations of alternative splicing events in the EC1 domain and IC3 loop have not yet been clarified. In this study, we have used semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to examine the tissue distributions of four PAC1 isoforms in mice. We then established cell lines constitutively expressing each of the PAC1 isoforms and characterized the binding properties of each isoform to PACAP-38, vasoactive intestinal polypeptide (VIP), and the PAC1-specific agonist maxadilan, as well as the resulting effects on two major intracellular signaling pathways: cAMP production and changes in the intracellular calcium concentration. The results demonstrate that the variants of the IC3 loop affect the binding affinity of the ligands for the receptor, whereas the variants of the EC1 domain primarily affect the intracellular signaling downstream of PAC1. Accordingly, this study indicates that the combination of alternative splicing events in the EC1 domain and the IC3 loop create a variety of PAC1 isoforms, which in turn may contribute to the functional pleiotropism of PACAP. This study not only contributes to the understanding of the multiple functions of PACAP but also helps to elucidate the relationship between the structures and functions of G-protein-coupled receptors.

Isolation of the bioactive peptides CCHamide-1 and CCHamide-2 from Drosophila and their putative role in appetite regulation as ligands for G protein-coupled receptors

There are many orphan G protein-coupled receptors (GPCRs) for which ligands have not yet been identified. One such GPCR is the bombesin receptor subtype 3 (BRS-3). BRS-3 plays a role in the onset of diabetes and obesity. GPCRs in invertebrates are similar to those in vertebrates. Two Drosophila GPCRs (CG30106 and CG14593) belong to the BRS-3 phylogenetic subgroup. Here, we succeeded to biochemically purify the endogenous ligands of Drosophila CG30106 and CG14593 from whole Drosophila homogenates using functional assays with the reverse pharmacological technique, and identified their primary amino acid sequences. The purified ligands had been termed CCHamide-1 and CCHamide-2, although structurally identical to the peptides recently predicted from the genomic sequence searching. In addition, our biochemical characterization demonstrated two N-terminal extended forms of CCHamide-2. When administered to blowflies, CCHamide-2 increased their feeding motivation. Our results demonstrated these peptides actually present as the major components to activate these receptors in living Drosophila. Studies on the effects of CCHamides will facilitate the search for BRS-3 ligands.

Orphan GPCRs and methods for identifying their ligands.

G protein-coupled receptors (GPCRs) constitute the largest family of cell-surface receptors. These proteins play a crucial role in physiology by facilitating cell communication through recognition of diverse ligands, including bioactive peptides, amines, nucleosides, and lipids. The human genome sequencing project identified more than 100 orphan GPCRs, whose ligands had not yet been discovered. We subsequently identified ghrelin, neuromedin U, and neuromedin S as endogenous ligands of various orphan GPCRs and have proposed various mechanisms through which these peptides regulate physiological functions through their cognate GPCRs. In this chapter, we review methods for identifying novel peptide ligands of orphan GPCRs.

Mitsugumin 53-mediated maintenance of K+ currents in cardiac myocytes.

Mitsugumin 53 (MG53) is a muscle-specific RBCC/TRIM family member predominantly localized on small vesicles underneath the plasma membrane. Upon cell-surface lesion MG53 recruits the vesicles to the repair site in an oxidation-dependent manner and MG53-knockout mice develop progressive myopathy associated with defective membrane repair. In this report, we focus on MG53-knockout cardiomyocytes showing abnormal action potential profile and a reduced K+ current density. In cDNA expression experiments using cultured cells, KV2.1-mediated currents were remarkably increased by MG53 without affecting the total and cell-surface levels of channel expression. In imaging analysis MG53 seemed to facilitate the mobility of KV2.1-containing endocytic vesicles with acidic pH. However, similar effects on the current density and vesicular mobility were not observed in the putative dominant-negative form of MG53. Our data suggest that MG53 is involved in a constitutive cycle of certain cell-surface proteins between the plasma membrane and endosome-like vesicles in striated muscle, and also imply that the vesicular dynamics are essential for the quality control of KV2.1 in cardiomyocytes.

Suppressive effects of dRYamides on feeding behavior of the blowfly, Phormia regina.

Recently, dRYamides-1 and -2 have been identified as ligands of the neuropeptide Y-like receptor CG5811 in Drosophila melanogaster. It has also been reported in brief that injection of dRYamide-1suppresses the early feeding behavior called proboscis extension reflex (PER) in the blowfly Phormia regina. Immunohistochemical analyses by our group using anti-dRYamide-1 antiserum indicated symmetrical localization of 32 immunoreactive cells in the brain of P. regina. In order to analyze the mechanism of feeding regulation, we further investigated the effects of dRYamide-1 and -2 on intake volume, PER exhibition, and activity of the sugar receptor neuron. After injection of dRYamide-1 or -2, flies showed little change in the intake volume of sucrose solution, but a significant depression of PER to sucrose. Injection of dRYamide-1 revealed a significant decrease in the responsiveness of the sugar receptor neuron, although the injection of dRYamide-2 did not. These results suggest that the dRYamide peptides decrease feeding motivation in flies, as evaluated by PER threshold, through a mechanism that partially involves desensitization of the sugar receptor neuron.

Alternative Splicing of the Pituitary Adenylate Cyclase-activating Polypetide (PACAP) Receptor Contributes to Function of PACAP-27

Pituitary adenylate cyclase-activating polypeptide (PACAP)-27 and PACAP-38 are neuropeptides performing a variety of physiological functions. The PACAP-specific receptor PAC1 has several variants that result mainly from alternative splicing in the mRNA region encoding the first extracellular (EC1) domain and the third intracellular cytoplasmic (IC3) loop. To characterize the molecular forms of alternative splicing variants of PAC1, we examined the binding affinity and activation of two major second messenger pathways (cAMP production and changes in [Ca2+]i) by PACAP-27. Activation of cAMP was influenced by the variant in both of the EC1 domain and IC3 loops. In the N form in the EC1 domain, the suppressive effect of the HOP1 form in the IC3 loop was enhanced. Regarding the intracellular calcium mobilization assay, the rank order of the potency of PACAP-27 for the different PAC1 isoforms was S/HOP1 >> N/R ≅ S/R >> N/HOP1. In particular, PACAP-27 exhibited remarkable potency of calcium mobilization in the S/HOP1-expressing cells at sub-picomolar concentrations even though the affinities of PACAP-27 to the four PAC1 isoforms were not significantly different. This suggests the specific functions of PACAP-27 due to PACAP-27 preferring PAC1 activation, and leads in clarification of the pleiotoropic function of PACAP.

Purification and characterization of bioactive peptides RYamide and CCHamide in the kuruma shrimp Marsupenaeus japonicus

To understand the regulation systems of appetite, bioactive peptides from the kuruma shrimp Marsupenaeus japonicus (Mj) were isolated and purified by reverse pharmacological assays using CHO cells expressing the Drosophila melanogaster G-protein-coupled receptors (GPCRs) CG5811 (a RYamide receptor) or CG14593 (a CCHamide-2 receptor). Four peptides having binding activity to GPCRs were obtained and named Mj RYamide-1, Mj RYamide-2, Mj RYamide-3, and Mj CCHamide. Genes encoding the prepropeptides of these peptides were identified using kuruma shrimp transcriptome databases. The Mj prepro-RYamide gene encodes a 130-amino acid polypeptide containing Mj RYamide-1, Mj RYamide-2, and Mj RYamide-3, whereas the Mj prepro-CCHamide gene encodes a 119-amino acid polypeptide containing a single Mj CCHamide peptide. The expression of these genes was confirmed in various neuronal organs including the brain and ventral nerve cord. In addition, prepro-RYamide gene expression is significantly reduced in the brain after starvation. RYamides may thus be associated with regulation of feeding or digestion. Changes in kayak (the c-fos ortholog in invertebrates) gene expression after administration of synthetic peptides were also investigated. Mj kayak expression levels are upregulated in hepatopancreas after treatment with Mj RYamide-3 or CCHamide. Thus, the peptides isolated in this study may have some regulatory effect on cellular metabolism in aquacultured invertebrates.

Conformational Change in the Ligand-Binding Pocket via a KISS1R Mutation (P147L) Leads to Isolated Gonadotropin-Releasing Hormone Deficiency

Context: Kisspeptin receptor (KISS1R) is expressed in hypothalamic gonadotropin-releasing hormone neurons and responsible for pubertal onset and reproductive functions. KISS1R mutations remain a rare cause of congenital hypogonadotropic hypogonadism (CHH). Objective: The aim of this study was to identify the genetic cause of CHH in a patient and to functionally characterize a KISS1R mutation. Design: The patient was a 47-year-old Japanese man whose parents were first cousins. He lacked secondary sexual characteristics owing to normosmic CHH. Exon segments for the KISS1R gene in this patient were screened for mutations. Functional analyses were performed using HEK293 cells expressing KISS1R mutants. Molecular dynamics simulations were performed to compare the ligand-KISS1R mutant complex with those of wild-type KISS1R variants. Results: A homozygous mutation (c.440C>T, p.P147L) in KISS1R was identified. The P147L mutation did not affect either receptor expression level or subcellular localization in the recombinant expression system. Intracellular calcium measurements and cellular dielectric spectroscopy demonstrated that the P147L mutation impaired receptor function to an extent more severe than that of a previously reported L148S mutation. A receptor-ligand binding assay showed the P147L mutation causes a substantial loss of ligand-binding affinity. Molecular dynamics simulations revealed the P147L mutation decreases the contact surface area of the ligand-receptor complex in an expanded ligand-binding pocket. Conclusion: We identified a loss-of-function mutation in KISS1R associated with CHH. Our results demonstrated that the P147L mutation causes a severe phenotype and functional impairment resulting from the loss of ligand-binding affinity due to an expanded ligand-binding pocket.