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)
Schematic diagram for the genomic and non-genomic actions of ecdysteroids (E). E can bind to nuclear receptors to control the expression of genetic information. E and dopamine (DA) can differentially couiple the cell surface GPCR DmDopEcR (CG18314) to different second messenger pathways and produce rapid non-genomic responses.
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
Updated 30 August, 2011