Putative leech dopamine1-like receptor molecular characterization: sequence homologies between dopamine and serotonin leech CNS receptors explain pharmacological cross-reactivities.
The biochemical characterization of a serotonin (5HT) receptor and the cloning of a dopamine (DA) receptor in the central nervous system (CNS) of the leech, Theromyzon tessulatum, is presented. Additionally, DA and 5HT binding sites were examined in the CNS by Scatchard analysis which showed a single, relatively high-affinity binding site with a Kd 1.1 nM and a Bmax 126+18 fmol/mg protein for [3H]DA and a Kd 2.1 nM and a Bmax 225 fmol/mg protein for [3H]5HT. The first 88 amino acids of the 5HT receptor, isolated by a 5HT-affinity column followed by anion exchange chromatography and C3 reverse-phase HPLC exhibited a 43% sequence homology with Lymnaea stagnalis 5HT-receptor. The isolated DA receptor revealed a single protein of 45 kDa with an anti-D1-R in Western blot. The first 80 N-terminal amino acid residues and a trypsin digested fragment of 31 residues were obtained, and based on these sequencing data, a molecular biology strategy using reverse transcriptase-polymerase chain reaction, was developed. An amplified 1-kb segment was obtained. The complete deduced sequence of 416 amino acid residues exhibited about 30.6% sequence homology with the vertebrate D1 receptor family. Moreover, we further demonstrate that the leech 5HT and DA receptors also exhibit 30% sequence identity with each other, explaining their pharmacological cross-reactivity. Finally, anti-D1-R immunocytochemistry revealed positive structures in the peripheral and central nervous system, e.g., neurons, sensory fibers and immune cells. This is the first biochemical and molecular characterization of a DA receptor in leeches.