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

Abstract

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.

Keywords: CCHamide; Drosophila; GPCR; bombesin receptor subtype 3; novel bioactive peptide.

FIGURE 1

Purification of CCHamide-1 from fly extracts. Black bars indicate changes of [Ca²⁺]_i fluorescence signal in CHO-CG30106 cells. (A) G-50 gel filtration of the SP-III fraction of fly extracts. The active fraction was subjected to one step of CM-ion-exchange HPLC and three steps of RP-HPLC. (B) Final purification of the active fraction by RP-HPLC. (C) Nucleotide sequence and deduced amino acid sequence of CCHamide-1 cDNA. CCHamide-1 cDNA encode 182-residue peptides. The asterisk indicates a glycine residue that serves as an amide donor for C-terminal amidation. The CCHamide-1 sequence is underlined as (1). (D) Chromatographic comparison by RP-HPLC of natural CCHamide-1 and synthetic CCHamide-1. Black bar (P1) indicates the changes of [Ca²⁺]_i fluorescence signal in CHO-CG30106 cells. Each peptide was applied to a Symmetry C18 column (3.9 mm × 150 mm, Waters, MA, USA) with a 10–60% ACN/0.1% trifluoroacetic acid (TFA) linear gradient at a flow rate of 1 ml/min for 80 min. P1 represent active fraction containing natural CCHamide-1. (a) Synthetic CCHamide-1. (E) Active fractions of each chromatography and the amino acid sequence of CCHamide-1.

FIGURE 2

Purification of CCHamide-2 from fly extracts. Black bars indicate changes of [Ca²⁺]_i fluorescent signal in CHO-CG14593 cells. (A) G-50 gel filtration of the SP-III fraction of fly extracts. The active fraction was subjected to one step of CM-ion-exchange HPLC and three steps of RP-HPLC. (B–D) Final purification of the active fraction by RP-HPLC. (E) Nucleotide sequence and deduced amino acid sequence of CCHamide-2 cDNA. CCHamide-2 cDNA encodes a 136-residue peptides. The asterisk indicates a glycine residue that serves as an amide donor for C-terminal amidation. The CCHamide-2 sequence is underlined as (4). The other long-form of CCHamide-2 is translated from (2) or (3). (F) Chromatographic comparison by RP-HPLC of natural CCHamide-2 and synthetic CCHamide-2. Black bars (P2, P3) indicate the changes of [Ca²⁺]_i fluorescence signal in CHO-CG14593 cells. Each peptide was applied to a Symmetry C18 column with a linear gradient elution for 80 min. P2 and P3 represent active fractions containing natural CCHamide-2. (b) Synthetic long-form of CCHamide-2. (c) Synthetic CCHamide-2. (G) Active fractions of each chromatography and the amino acid sequence of CCHamide-2.

FIGURE 3

Pharmacological characterization of synthetic peptides using CG30106 or CG14593 stably expressed in CHO cells. (A,B) Concentration–response relationships of changes in [Ca²⁺]_i for CCHamide-1 (open circle) and CCHamide-2 (open square), in CHO-CG30106 cells (A) or CHO-CG14593 cells (B). (C,D) Concentration–response relationships of changes in [Ca²⁺]_i for various peptides, CCHamide-1 (open circle), and CCHamide-2 (open square) in CHO-CG30106 cells (C) or CHO-CG14593 cells (D). Non-C-terminal amidated CCHamide-1 (filled circle), non-C-terminal amidated CCHamide-2 (filled square), long-form CCHamide-2 (open triangle), and non-C-terminal amidated long-form CCHamide-2 (filled triangle). Each symbol on the line graph represents the mean ± SEM of data from six replicates for each experiment.

FIGURE 4

Effect of CCHamide-2 on PER of the blowfly. Sigmoidal curves show the sucrose concentration–PER relationship for three fly groups: no injection (closed circle), injection with linger solution (open square), and injection of CCHamide-2 (open triangle). Each symbol on the line graph represents the mean ± SEM of data from five replicates for each experiment.