The biogenic amine 5-hydroxytryptamine (5-HT) acts as a chemical messenger in most animal phyla in which it controls and modulates a great variety of important physiological and behavioural processes (Weiger, 1997). Disruption of the 5-hydroxytryptaminergic system has been linked to several human disease states, such as schizophrenia, migraine, depression, suicidal behaviour, infantile autism, eating disorders and obsessive–compulsive disorder (Jones and Blackburn, 2002). In insects, 5-HT signalling has been ascribed as being involved in the modulation of the heart rate (Zornik et al., 1999), secretory processes (Just and Walz, 1996), development (Colas et al., 1999), circadian rhythms (Page, 1987; Yuan et al., 2005), aggression (Dierick and Greenspan, 2007), behavioural gregarization in locusts (Anstey et al., 2009) and learning and memory (Sitaraman et al., 2008). The multifaceted actions of 5-HT are mediated by binding to integral membrane receptors, most of which, except for the 5-HT3 receptor ion channel, belong to the superfamily of G-protein-coupled receptors (GPCRs). In vertebrates, six main classes of G-protein-coupled 5-HT receptors have been classified on the basis of their sequence similarities, gene organization, second-messenger coupling pathways and pharmacological characteristics (Hoyer et al., 2002; nomenclature follows Alexander et al., 2009). The 5-HT1 and 5-HT5 receptors couple preferentially to Gi/o proteins, and inhibit cAMP synthesis. The 5-HT2 receptors couple preferentially to Gq/11 proteins, which mediate the hydrolysis of inositol phosphates and a subsequent increase in cytosolic Ca2+ levels. The 5-HT4, 5-HT6 and 5-HT7 receptors all couple preferentially to Gs proteins, and promote cAMP formation. In invertebrates, the 5-hydroxytryptaminergic system might be similarly complex (Hauser et al., 2006). For example, the fruit fly Drosophila melanogaster is known to express at least four 5-HT receptor subtypes that are predicted to be orthologs of the mammalian 5-HT1A, 5-HT2 and 5-HT7 receptors. These are the Dm5-HT1A and Dm5-HT1B (Saudou et al., 1992), Dm5-HT2 (Colas et al., 1995) and Dm5-HT7 (Witz et al., 1990) receptors, respectively (see Nichols, 2006; Nichols and Nichols, 2008). The sequences and the signal transduction mechanisms of 5-HT receptors are generally highly conserved between vertebrates and invertebrates (Hen, 1992). In contrast, the pharmacological properties of vertebrate and invertebrate receptors vary significantly in many cases (see Blenau and Baumann, 2001; Tierney, 2001). The 5-HT1 receptors form the largest class of 5-HT receptors (Gerhardt and van Heerikhuizen, 1997). In vertebrates, several members of this class show agonist-independent activation of associated G proteins, and thus are constitutively active (McLoughlin and Strange, 2000; Martel et al., 2007). Constitutive activity is now accepted as being a common property of many GPCRs (Seifert and Wenzel-Seifert, 2002), but has not, as yet, been shown for any insect 5-HT1 receptor.
In the present study, we have cloned and characterized a 5-HT receptor of the cockroach Periplaneta americana with significant homologies to members of the 5-HT1 receptor class. Cockroaches have been widely used as a model organism for basic research in physiology and neurobiology (Downer, 1990; Watanabe and Mizunami, 2007). In particular, the salivary gland of P. americana is a well-established model system for studying excitation–secretion coupling in epithelia and aminergic signal transduction (see House and Ginsborg, 1985; Walz et al., 2006). Information has thus been accumulated on the pharmacology of amine receptors in the salivary glands and other tissues of cockroaches (Downer, 1990; Walz et al., 2006; Troppmann et al., 2007). Comparatively little is known, however, concerning the exact repertoire and molecular properties of amine receptors in P. americana (Bischof and Enan, 2004; Rotte et al., 2009), and, until this study, no molecular data on 5-HT receptors have been available.
In this investigation, we show that the mRNA encoding a cockroach 5-HT1 receptor is expressed in the brain, salivary gland and midgut tissue. Immunohistochemical analysis has revealed the presence of the receptor protein in a specific subset of pars intercerebralis cells of the cockroach brain. When stably expressed in HEK 293 cells, the receptor inhibits the formation of cAMP with an EC50 of ∼130 nM for serotonin. The receptor shows constitutive activity, which can be blocked by the 5-HT1A receptor antagonist WAY 100635. Our study has therefore elucidated unique molecular and pharmacological details of an insect 5-HT1 receptor, and advances our knowledge concerning the complexity of the 5-hydroxytryptaminergic system in insects.