Functional characterization and expression analysis of the myoinhibiting peptide receptor in the Chagas disease vector, Rhodnius prolixus

Paluzzi JP, Haddad AS, Sedra L, Orchard I, Lange AB

Mol. Cell. Endocrinol. 2015 Jan;399:143-53

PMID: 25218475

Abstract

Myoinhibiting peptides (MIPs), which are also known as B-type allatostatins, are a family of neuropeptides found in protostomes. Their primary structure is characterized by an amidated carboxyl-terminal motif consisting of a conserved pair of tryptophan residues normally separated by six non-conserved amino acids (W(X6)Wamide). In the fruit fly Drosophila melanogaster, MIPs are likely the ancestral ligands of the sex peptide receptor, which plays an important role in courtship and reproduction. Recently, several endogenous MIPs were discovered in the Chagas disease vector, Rhodnius prolixus, having both conserved (W(X6)Wamide) and atypical (W(X7)Wamide) carboxyl-terminal motifs. Physiological functions of MIPs are plentiful and include inhibition of visceral muscle activity; a role that has been illustrated on hindgut in R. prolixus. In order to identify novel physiological targets and elucidate biological actions for the MIPs in R. prolixus, we have isolated and examined the spatial expression profile of the MIP receptor transcript in various fifth instar tissues and have additionally determined the expression profile in reproductive tissues of fifth instar as well as adult insects. The most abundant MIP receptor transcript expression was found in the salivary glands and central nervous system, which corroborates roles previously determined for MIPs in other insects. We functionally-characterized the endogenous MIP receptor and examined its activation by R. prolixus MIPs containing the typical W(X6)Wamide and atypical W(X7)Wamide carboxyl-terminal motifs. These peptides dose-dependently activated the MIP receptor (RhoprMIPr1) with EC50 values in the mid-nanomolar range. We also examined the activity of these RhoprMIPs on spontaneous muscle contractions of oviducts from female adult R. prolixus. Our findings confirm the myoinhibitory nature of the MIP peptides, which dose-dependently reduced spontaneous oviduct contractions by nearly 70%, again having mid-nanomolar EC50 values. Finally, we utilized a heterologous receptor assay and oviduct bioassay to examine the activity of several MIP structural analogs, which independently confirmed the requirement of the highly conserved tryptophan residues as well as the amidated C-terminus for retaining full biological activity.

Myoinhibitors controlling oviduct contraction within the female blood-gorging insect, Rhodnius prolixus

Sedra L, Haddad AS, Lange AB

Gen. Comp. Endocrinol. 2015 Jan;211:62-8

PMID: 25482544

Abstract

Muscle activity can be regulated by stimulatory and inhibitory neuropeptides allowing for contraction and relaxation. There are various families of neuropeptides that can be classified as inhibitors of insect muscle contraction. This study focuses on Rhodnius prolixus and three neuropeptide families that have been shown to be myoinhibitors in insects: A-type allatostatins, myoinhibiting peptides (B-type allatostatins) and myosuppressins. FGLa/AST-like immunoreactive axons and blebs were found on the anterior of the dorsal vessel and on the abdominal nerves. FGLa/AST-like immunoreactive axons were also seen in the trunk nerves and on the bursa. The effects of RhoprAST-2 (FGLa/AST or A-type allatostatins) and RhoprMIP-4 (MIP/AST or B-type allatostatins) were similar, producing dose-dependent inhibition of R. prolixus spontaneous oviduct contractions with a maximum of 70% inhibition and an EC50 at approximately 10(-8)M. The myosuppressin of R. prolixus (RhoprMS) has an unusual FMRFamide C-terminal motif (pQDIDHVFMRFa) as compared to myosuppressins from other insects. Quantitative PCR results show that the RhoprMS receptor transcript is present in adult female oviducts; however, RhoprMS does not have an inhibitory effect on R. prolixus oviduct contractions, but does have a dose-dependent inhibitory effect on the spontaneous contraction of Locusta migratoria oviducts. SchistoFLRFamide, the myosuppressin of Schistocerca gregaria and L. migratoria, also does not inhibit R. prolixus oviduct contractions. This implies that FGLa/ASTs and MIP/ASTs may play a role in regulating egg movement within the oviducts, and that the myosuppressin although myoinhibitory on other muscles in R. prolixus, does not inhibit the contractions of R. prolixus oviducts and may play another role in the reproductive system.

Identification, characterization and expression of a receptor for the unusual myosuppressin in the blood-feeding bug, Rhodnius prolixus

Lee D, James TE, Lange AB

Insect Mol. Biol. 2015 Feb;24(1):129-37

PMID: 25318350

Abstract

Myosuppressins are a family of the FMRFamide-like peptides. They have been characterized in many insects and shown to inhibit visceral muscle contraction. Rhodnius prolixus possesses an unusual myosuppressin in that the typical FLRFamide C-terminal motif is unique and ends with FMRFamide. In the present study, we isolated the cDNA sequence for the R. prolixus receptor for this unusual myosuppressin (RhoprMSR). Quantitative PCR indicates high relative transcript expression of RhoprMSR in the central nervous system and also supports the previously described physiological effects of RhoprMS on the digestive system, with expression of the RhoprMSR transcript in the midgut and hindgut. Expression of the RhoprMSR transcript was also found in the female and male reproductive system of 5th instar nymphs, with transcript expression greater in the female reproductive tissues. No expression was found in the salivary glands or Malpighian tubules. A functional receptor expression assay confirmed that the cloned RhoprMSR is indeed activated by RhoprMS (half maximum effective concentration = 42.7 nM). Structure-activity studies based upon both functional receptor assays and physiological assays showed the importance of the HVFMRFamide moiety, as further N-terminal truncation removed all activity.