Structure–activity studies with endogenous allatostatins from Periplaneta americana: Expressed receptor compared with functional bioassay
Introduction
Allatostatins (ASTs) are neuropeptides that were originally isolated from the brain of the cockroach Diploptera punctata and defined by their ability to inhibit the biosynthesis of juvenile hormone (JH) by corpora allata in vitro (Woodhead et al., 1989; Pratt et al., 1989). The allatostatin precursor of the cockroach Periplaneta americana contains 14 distinct allatostatin sequences (Peram-AST 1–14), ranging in length from 6 to 18 amino acids (Ding et al., 1995). The presence of a dibasic cleavage site within the longest peptide (Peram-AST 2) can give rise to an additional allatostatin, Peram-AST 211–18 (LPVYNFGLa) (where a=amide), raising the endogenous peptide total to 15. With the exception of Peram-AST 14 (where the C-terminal amino acid is isoleucine), each of the Peram-ASTs contains a variant of the conserved pentapeptide Y/FXFGLa (where X=D, N, G, S or A) at the C-terminus. This “message” sequence is required for biological activity of this peptide family (Stay et al., 1994; Pratt et al., 1997; Weaver et al., 1998; Tobe et al., 2000, Gäde, 2002).
Although only two allatostatins (Peram-AST 7; SPSGMQRLYGFGLa and Peram-AST 9; ADGRLYAFGLa) were originally isolated from the brain/retrocerebral complex of P. americana (Weaver et al., 1994), mass spectrometric studies have demonstrated 11 of the allatostatins in the retrocerebral complex of P. americana, and 13 in the antennal pulsatile organ (Predel et al., 1999; Predel, 2001).
Originally defined as inhibitors of JH biosynthesis in corpora allata of cockroaches, the A-type allatostatins have since been implicated in a variety of functional roles, and may not be directly involved in the regulation of JH biosynthesis in most insect species. The apparent diversity of function encompasses suppression of muscular activity of different tissues (e.g. foregut, hindgut, antennal pulsatile organ, oviduct and heart), inhibition of vitellogenesis and modulation of the activity of certain digestive enzymes from the midgut activity (review by Gäde, 2002). The level of response of different tissues in different species may vary, and may also show a rank order of efficacy that differs from that of allatostatic activity (Lange et al., 1995; Tobe et al., 2000). Similarly, differences have been reported for the level of JH-inhibitory response to different allatostatins during different stages of the oocyte development cycle (Pratt et al., 1997). These observations, coupled with the multiplicity of allatostatins in all species examined, point to the existence of distinct allatostatin receptor sub-types, possible functional differences in allatostatin ligand-receptor interaction, or perhaps functional redundancy of some of the allatostatin peptides.
Using primarily PCR-based approaches, Birgül et al. (1999) and Lenz et al., 2000a, Lenz et al., 2000b characterised two G-protein-coupled receptors from Drosophila melanogaster (named DAR-1 and -2) that are structurally related to mammalian galanin receptors and which appear to be receptors for allatostatin in D. melanogaster. Using a similar approach, Auerswald et al. (2001) identified a related allatostatin receptor (Peram-AlstR) from the cockroach P. americana, which was functionally characterised using a limited number of D. punctata allatostatins. A further allatostatin receptor (BAR) has been identified from the silkworm Bombyx mori (Secher et al., 2001) and again tested for ligand activation by a limited range of allatostatins.
To obtain a better understanding of ligand–receptor interactions of endogenous allatostatins in relation to biological activity in the American cockroach, we report structure–activity studies of all 15 endogenous Peram-ASTs, and some analogues, using a cloned and expressed Peram-AlstR, and compare these data to measurements of inhibition of JH biosynthesis in vitro. We report also on the spatial expression of the Peram-AlstR in adult P. americana by RT-PCR methods, and show that the receptor is expressed in early instars.
Section snippets
Functional expression of allatostatin receptor in Xenopus oocytes
The cDNA for the P. americana allatostatin receptor (Peram-AlstR) was sub-cloned into the Xenopus expression vector pGEMHE, which contains the 5′- and 3′-untranslated regions of the Xenopus laevis globin. Likewise, the cDNA for the mouse G-protein gated inwardly rectifying potassium channel sub-unit (GIRK1) was cloned into the mammalian expression vector pcDNA3. Co-expression of the Peram-AlstR and GIRK1 cRNAs in oocytes of X. laevis and whole-cell voltage-clamp recordings were performed as
Dose–response activation of the Peram-AlstR receptor by endogenous allatostatins and analogues
Allatostatin receptor cRNA from P. americana was co-injected with cRNA coding for the mouse GIRK1 into X. laevis oocytes, and these oocytes were exposed to a range of doses of endogenous Peram-ASTs and analogues in high potassium medium. Each of the 15 Peram-ASTs encoded by the P. americana allatostatin prohormone gene, including the C-terminal octapeptide amide processing fragment of Peram-AST 2 (Peram-AST 211–18) induced strong dose-dependent inward currents in oocytes in which the receptor
Dose–response relationships of the P. americana allatostatins
This is the first report of the functional assessment of a complete range of endogenous A-type allatostatins against a cloned cognate A-type allatostatin receptor. The EC50 values show a wide variation in potency between the different homologues. Similar variations in potency have been reported for other homologous peptides from single prohormone genes, when tested against their respective cloned receptors. Examples include, the D. melanogaster allatostatin receptors, DAR-1 and DAR-2 (Larsen et
Acknowledgements
G.G. is indebted to the German Academic Exchange Service (DAAD, Bonn, Germany) for financial support during his 3 months stay in Hamburg, to the National Research Foundation (Pretoria, South Africa; Grant number 2053396) and the University of Cape Town for financial support during his sabbatical. HGM wishes to acknowledge financial support via a prestigious fellowship granted by the Alexander von Humboldt Foundation (Bonn, Germany). R.J.W. acknowledges support from the Pesticides Safety
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