Arthropod 5-HT₂Receptors: A Neurohormonal Receptor in Decapod Crustaceans That Displays Agonist Independent Activity Resulting from an Evolutionary Alteration to the DRY Motif

Abstract

The stomatogastric nervous system (STNS) is a premiere model for studying modulation of motor pattern generation. Whereas the cellular and network responses to monoamines have been particularly well characterized electrophysiologically, the transduction mechanisms that link the different monoaminergic signals to specific intracellular responses are presently unknown in this system. To begin to elucidate monoaminergic signal transduction in pyloric neurons, we used a bioinformatics approach to predict the existence of 18 monoamine receptors in arthropods, 9 of which have been previously cloned in Drosophila and other insects. We then went on to use the two existing insect databases to clone and characterize the 10th putative arthropod receptor from the spiny lobster, Panulirus interruptus. This receptor is most homologous to the 5-HT 2 subtype and shows a dose-dependent response to 5-HT but not to any of the other monoamines present in the STNS. Through a series of pharmacological experiments, we demonstrate that this newly described receptor, 5-HT 2␤Pan , couples with the traditional G q pathway when expressed in HEK293 cells, but not to G s or G i/o . Moreover, it is constitutively active, because the highly conserved DRY motif in transmembrane region 3 has evolved into DRF. Site-directed mutagenesis that reverts the motif back to DRY abolishes this agonist-independent activity. We further demonstrate that this receptor most likely participates in the modulation of stomatogastric motor output, because it is found in neurites in the synaptic neuropil of the stomatogastric ganglion as well as in the axon terminals at identified pyloric neuromuscular junctions.

Arthropod D2 receptors positively couple with cAMP through the Gi/o protein family

“…The lobster D 2αPan cDNA was cloned from nervous tissue of Panulirus interruptus using a degenerate PCR strategy with conventional library screening and RACE technology as previously described (Clark et al, 2004). The D 2α.1Pan sequence has been submitted to Genbank under accession number DQ900655 (Figure 1).…”

“…Biosciences Clontech, Palo Alto, CA) using standard recombinant techniques. D 2αPan and AmDop3 constructs were stably expressed in HEK293 cells using methods previously described (Clark et al, 2004). AmDop3 was kindly provided by Dr. Allison Mercer, University of Otago.…”

“…cAMP levels were measured as previously described (Clark et al, 2004). Briefly, 1 × 10 5 cells were plated in 35mm dishes and grown to confluence.…”

“…Data mining studies suggest that there are roughly 19 monoamine receptors in arthropods (Clark et al, 2004;Roeder, 2003). By the year 2004, 10 of these receptors had been cloned and characterized (Tierney, 2001;Blenau and Baumann, 2001;Clark et al, 2004).…”

“…Data mining studies suggest that there are roughly 19 monoamine receptors in arthropods (Clark et al, 2004;Roeder, 2003). By the year 2004, 10 of these receptors had been cloned and characterized (Tierney, 2001;Blenau and Baumann, 2001;Clark et al, 2004). Several recent efforts have reduced the number of uncharacterized monoaminergic receptors to roughly 3 out of 19 (Balfanz et al, 2005;Srivastava et al, 2005;Maqueira et al, 2005;Cazzamali et al, 2005;Evans and Maqueira, 2005).…”