Peter Evans
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• Recent, selected Publications

Rapid non-genomic actions of steroids mediated through cell surface G-protein coupled receptors

Cell surface receptors convey information about the extracellular environment of cells to intracellular pathways that can respond appropriately to messages, or changes, in this environment. Our goal is to increase knowledge of the molecular basis of signalling through such receptors. We are focusing our research on signalling mechanisms through 7-Transmembrane spanning (7-TM) G-protein coupled receptors. Such receptors are important target sites for the actions of many clinically important drugs and are also increasingly being recognised as the sites of genetic defects underlying diseases. Our aims are to identify novel molecules involved in cell to cell signalling, to clone and sequence the receptors for such molecules and to determine their coupling abilities to second messenger systems. We are particularly interested in the mechanisms and processes whereby such receptor mediated signalling can be modified by extracellular and intracellular pathways.

We are currently focussing our research on a novel group of Drosophila G-protein coupled receptors that have a structural homology with vertebrate β-adrenergic receptors. One receptor from this group, DmDopEcR (CG18314), can be activated by both the catecholamine, dopamine, and by insect ecdysteroids. When this receptor is expressed either in a Chinese hamster ovary cell line or via a baculovirus expression system in the insect Sf9 cell line, it can be coupled to the stimulation of adenylyl cyclase activity by exposure to dopamine. Dopamine can also activate the PI3 Kinase pathway through this receptor by both a rapid and dose-dependent pathway as assessed by Akt phosphorylation. The pharmacology of this response is unusual since it does not correspond to any of the known pharmacological profiles for dopaminergic or adrenergic receptor subtypes. However, unusual adrenergic receptor pharmacology has been described in vertebrate brain, blood vessels and pancreas where the receptor responds equally to dopamine, noradrenaline and adrenaline. The receptors underlying these responses, although not yet cloned, have been designated as “γ-adrenergic receptors”. Interestingly, these receptors have recently also been suggested to underlie some of the rapid, non-genomic responses of pancreatic cells to the vertebrate steroid, 17β-estradiol. GPCRs may also play important roles in the production of rapid non-genomic effects for a range of other vertebrate steroids.

Thus, we have examined whether the insect ecdysteroids, ecdysone and 20-Hydroxyecdysone can also activate the DmDopEcR receptor. The receptor shows specific binding and a high affinity for the plant edysteroid, ponasterone, in membrane preparations from Sf9 cells. This binding can be displaced by ecdysone more easily than by 20-hydroxyecdysone. This, and other observations, suggests that the pharmacology of this receptor is very different from that of the conventional nuclear receptor which mediates the genomic effects of ecdysteroids on insect development. DmDopEcR has a much higher affinity for ecdysteroids than it does for the catecholamines and they can block the stimulatory actions of dopamine on the receptor. In addition, stimulation of the receptor by ecdysteroids alone selectively activates the MAPKinase pathway, as assessed by ERK1/2 phosphorylation, in both a rapid (maximum response: 15 minutes) and a dose-dependent manner that does not depend on protein synthesis.

Figure 1 (Click to enlarge)

Hence some of the rapid, non-genomic actions of ecdysteroids during insect development and in the modulation of adult neuronal activity might be mediated via the activation of the cell surface GPCR, DmDopEcR (Figure 1). Ecdysteroids are released in a pulsatile fashion during insect development and thus this receptor may function as a molecular switch during development to turn off signalling through this receptor by dopamine and to activate alternative pathways appropriate for that stage of development after ecdysteroid activation.




Recent, selected publications

Burman C, Maqueira B, Coadwell WJ, Evans PD (2007) Eleven new putative aminergic G-protein coupled receptors from Amphioxus (Branchiostoma floridae): identification, sequence analysis and phylogenetic relationship.
Invertebrate Neuroscience 7 87-98
http://dx.doi.org/10.1007/s10158-006-0041-z

Dossey AT, Reale V, Chatwin H, Zachariah C, deBono M, Evans PD, Edison AS (2006) NMR analysis of Caenorhabditis elegans FLP-18 neuropeptides: implications for NPR-1 activation.
Biochemistry 45 7586-7597
http://dx.doi.org/10.1021/bi0603928

Evans PD, Maqueira B (2005) Insect octopamine receptors: a new classification scheme based on studies of cloned Drosophila G-protein coupled receptors.
Invertebrate Neuroscience 5 111-118
http://dx.doi.org/10.1007/s10158-005-0001-z

Maqueira B, Chatwin HM, Evans PD (2005) Identification and characterization of a novel family of Drosophila β-adrenergic-like octopamine G-protein coupled receptors.
Journal of Neurochemistry 94 547-560
http://dx.doi.org/10.1111/j.1471-4159.2005.03251.x

Srivastava DP, Yu EJ, Kennedy K, Chatwin HM, Reale V, Hamon M, Smith TS, Evans PD (2005) Rapid, nongenomic responses to ecdysteroids and catecholamines mediated by a novel Drosophila G-protein-coupled receptor.
Journal of Neuroscience 25 6145-6155
http://dx.doi.org/10.1523/JNEUROSCI.1005-05.2005

Reale V, Chatwin HM, Evans PD (2004) The activation of G-protein gated inwardly rectifying K+ channels by a cloned Drosophila melanogaster neuropeptide F-like receptor.
European Journal of Neuroscience 19 570-576
http://dx.doi.org/10.1111/j.0953-816X.2003.03141.x

Feng G, Reale V, Chatwin HM, Kennedy K, Venard R, Ericsson C, Yu K, Evans PD, Hall LM (2003) Functional characterization of a neuropeptide F-like receptor from Drosophila melanogaster.
European Journal of Neuroscience 18 227-238
http://dx.doi.org/10.1046/j.1460-9568.2003.02719.x

Rogers C, Reale V, Kim K, Chatwin HM, Li C, Evans PD, De Bono M (2003) Inhibition of Caenorhabditis elegans social feeding by FMRFamide-related peptide activation of NPR-1.
Nature Neuroscience 6 1178-1185
http://dx.doi.org/10.1038/nn1140

Rudling JE, Richardson JE, Evans PD (2000) A comparison of agonist-specific coupling of cloned human α2-adrenoceptor subtypes.
British Journal of Pharmacology 131 933-941
http://dx.doi.org/10.1038/sj.bjp.0703644