When Pfizer's wonder drug Viagra hit the market, the press went wild and cyclic guanine monophosphate (cGMP) hit the big time. By preventing breakdown of the tiny signalling molecule, the drug alters a cell signalling pathway that governs vascular flow, with spectacular consequences for some tissues. But cGMP amplifies cellular messages in many other cell types and tissues, by activating two different protein kinases to trigger a wide variety of responses. Although research into cGMP signalling in cell culture has taught us much about many of the signal's targets, little is currently known about the physiological consequences of many of these signalling cascades in whole tissue. However, Shireen Davies and Julian Dow working in Glasgow, Scotland,are keen to reverse this trend. By focusing on cGMP signalling in the humble fruit fly, they hope to integrate intricate molecular signalling pathways with the tiny insect's physiology to discover the true physiological consequences of some of these complex signalling pathways.
Knowing that Malpighian tubule secretion is a highly regulated process,Davies and Dow have been able to use the tissue to identify key components of cGMP signalling systems in a physiological context. In a paper published in the current issue of the J. Exp. Biol., they have turned their attention to a signalling system that was first identified in the heads of mutated drosophila larvae, where the mutation affects a protein kinase known as DG2, to identify components of the signalling cascade(p. 2769).
Davies explains that insects carrying the dg2 mutation have an unusual behavioural pattern. Mutant flies tend not to forage when food is available and are known as having the forS phenotype, while wild type flies roam freely. Curious to know whether the mutation affected the insect's Malpighian tubules, Matthew MacPherson, Kate Broderick and Davies began testing the insect's Malpighian tubules to see if secretion was affected by the dg2 mutation, and found that wild-type flies had normal secretion rates when stimulated with the neurohormone capa-1, yet mutant flies had a much greater secretory response when treated with capa-1.
But which component of the cGMP cascade was altered by the dg2mutation? Knowing that the mutation appeared to decrease DG2's kinase activity in the heads of mutant flies, Davies suspected that the mutant DG2 protein was less active than in wild-type flies. But when she tested the kinase activity,amazingly it was unchanged. So the modified protein must be targeting another component of the signalling system to alter the Malpighian tubule's secretory properties.
Davies turned her attention to cGMP phosphodiesterase, which degrades cGMP and is a key signal regulator. The team measured the enzyme's activity to see if it was altered by the mutation in DG2. Davies admits that she was surprised when the phosphodiesterase activity decreased in the presence of capa-1. So instead of mediating its effect directly through the kinase, the dg2mutation regulates cGMP levels indirectly, by reducing the phosphodiesterase activity and subsequently lowering cGMP levels in the tissue to change the tubule's secretory properties.
Davies is delighted that this work has `explained the forSphenotype in tubules' and adds that this is the first example of neurohormone modulation of cGMP phosphodiesterase activity in insects, which could one day prove to be a novel target for insecticides.