Guilin Ren:
Functional characterization of CCHamide and muscarinic acetylcholine receptor signalling in Drosophila melanogaster

Date: 28-05-2015    Supervisor: Cornelis J.P. Grimmelikhuijzen

G-protein coupled receptors (GPCRs) constitute a large and ancient superfamily of membrane proteins responsible for the transduction of extracellular signals to the inside of the cells. In this Ph.D. thesis, Drosophila melanogaster (Dm) was used as a model organism to investigate a number of topics concerning the pharmacological and the physiological functions of GPCR signalling. The primary research in this Ph.D. thesis concerns two topics: (1) functional characterization of CCHamide-2 signalling and (2) functional characterization of muscarinic acetylcholine receptor (mAChR) signalling.

CCHamide-2 is a newly discovered insect peptide hormone. The function of this novel peptide has not been well characterised. In this Ph.D. thesis, I identified CCHamide-2 peptides in endocrine cells of the gut and neurones of the brain of larvae and endocrine cells of the gut of adult Drosophila. Behavioural assays in mutants created with the CRISP/Cas9 technique showed that CCHamide-2 is probly an orexigenic peptide and also that is an important factor for larval developmental timing.

In mammals, muscarinic acetylcholine signalling is involved in the signal transmission of the parasympathetic nervous system. However little is known about muscarinic acetylcholine receptor signalling in insects. In this study, I found that two types of mAChRs occur in D. melanogaster, one coupling to Gq (A-type) and the other to Gi (B-type). Both A- and B-type Dm-mAChRs can be activated by acetylcholine (ACh), but the classical antagonists atropine, scopolamine, and 3- Quinuclidinyl benzilate (QNB) can only block the A-type Dm-mAChRs, while it does not block Btype Dm-mAChRs. Furthermore, the sensitivity to muscarine is 1000× lower in B-type than in Atype Dm-mAChRs. By comparing the intracellular loops-2 and loops-3 of the D. melanogaster, Caenorhabditis elegans, and human mAChRs, I could identify hallmarks for Gi and Gq coupling. Appling these hallmarks to other mAChRs from animals with a sequenced genome, I could predict whether these receptors were Gq and Gi-coupled. In this way, I found that probly all animals have at least one Gq-coupled and one Gi-coupled mAChR.