Molecular cloning and heterologous expression of an α‐adrenergic‐like octopamine receptor from the silkworm Bombyx mori

Abstract

A cDNA encoding an octopamine (OA) receptor (BmOAR1) was isolated from the nerve tissue of silkworm (Bombyx mori) larvae. Comparison of amino acid sequences showed that BmOAR1 is highly identical to OA receptors isolated from Periplaneta americana (Pa oa(1)), Apis mellifera (AmOA1), and Drosophila melanogaster (OAMB or DmOA1A). BmOAR1 was stably expressed in HEK-293 cells. OA above 1 microM led to an increase in intracellular cyclic AMP concentration ([cAMP](i)). The synthetic OA-receptor agonist demethylchlordimeform also elevated [cAMP](i) to the same maximal level (approximately 5-fold over the basal level) as that induced by OA. However, other biogenic amines, tyramine and dopamine, and chlordimeform were without effects. The [cAMP](i) level raised by OA was lowered by antagonists; the rank order of antagonist activity was chlorpromazine > mianserin = yohimbine. Cyproheptadine and metoclopramide had little effect. OA above 100 nM induced a transient or sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), depending on the concentration of OA. Sequence homology and functional analysis data indicate that BmOAR1 is an alpha-adrenergic-like OA receptor of B. mori.

Review of octopamine in insect nervous systems

“…octopamine, inhibits adenylyl cyclase activity via coupling to inhibitory G proteins, inducing a decrease in intracellular cAMP levels ( Figure 7). 134,139,141,145,148 However, in other preparations, octopamine is more or equally as effective as tyramine in increasing intracellular Ca 2+ release. 135,136 These findings suggest that cloned receptors when expressed in different cell lines may be coupled with multiple effector pathways involving different G proteins.…”

“…These receptors exert a higher affinity for OCT than tyramine and are coupled with an increase in intracellular Ca 2+ concentration as well as a small increase in intracellular cAMP levels. 137,138,141,143,145 The OCTβ-R class shows sequence similarities with vertebrate β-adrenergic receptors, and activation of receptors in response to OCT specifically results in increased intracellular cAMP levels. 144,149 The OCTβ-R class is subdivided into several subclasses, which are pharmacologically different from each other.…”

“…26,[131][132][133][134][135][136][137][138][139][140][141][142][143][144][145][146][147][148] Based on the structural and signaling similarities between cloned D. melanogaster octopaminergic receptors and vertebrate adrenergic receptors, Evans and Maqueira proposed a new classification. 149 According to this new classification ( Figure 4B), octopaminergic receptors were grouped into three classes, ie, α-adrenergic-like (OCTα-R), β-adrenergic-like (OCTβ-R), and octopaminergic/tyraminergic (OCT/TYR-R) or tyraminergic (TYR-R).…”

“…150,151 Furthermore, the author has created a phylogenetic tree based on the comparison of 25 complete nucleotide sequences of insect octopaminergic and tyraminergic receptor genes by using the "Muscle" sequence alignment program. [153][154][155] Based on nucleotide sequence homology in the phylogenetic tree, insect octopaminergic receptor sequences from the moth M. sexta (MsOA), 146 D. melanogaster (OAMB) 137 and splice variants (DmOA1A, and DmOA1B), 143 A. mellifera (AmOA1), 138 P. americana (PaOA1), 142 B. mori (BmOAR1), 145 and locust Schistocerca gregaria (SgOctαR) 26 cluster together in the Octα-R class, whereas S. gregaria (SgOctβR), 26 B. mori (BmOAR2), 152 and D. melanogaster (DmOctβ1-R, DmOctβ2-R, DmOctβ3-R) 144 fall into the Octβ-R class ( Figure 5). Tyraminergic receptor sequences from A. mellifera (Amtyr1), 148 B. mori (BmTAR1), 140 (HvTA), 134 Agrotis ipsilon (Aityr/OCT), 141 Locusta migratoria (Lmtyr1), 139 and the cattle tick Boophilus microplus (BmiTA) 147 fall into the Tyr1-R subclass.…”

Bombyx mori phenolamine receptors: a comparative molecular biological study

“…8) A cDNA fragment was amplified by RT-PCR using primers designed based on the known sequences of Drosophila and Limnaea OA receptors, and 3Ј and 5Ј end sequences were obtained by RACE. The obtained full-length cDNA was found to encode a protein that contains seven transmembrane domains (TMs) and conserved amino acids characteristic of biogenic amine receptors.…”