Novel GPS-containing G protein-coupled receptor from Monosiga brevicollis

Abstract

Monosiga brevicollis is classified into the class Choanoflagellate (collar flagellates). These unicellular eukaryotes show the closest phylogenetic relationship to the metazoans. Most studies of choanoflagellates are performed in attempt to solve the key problem of evolution—how the transition from unicellular to multicellular organisms occurred. Analysis of M. brevicollis RNA and DNA showed that choanoflagellates, in contrast to other unicellular organisms, can express proteins that are homologous to proteins of metazoans (in particular, cadherins, C-type lectins, G protein-coupled receptors, and components of tyrosine kinase signaling cascade) [1]. In addition, it was shown that M. brevicollis genome contains immunoglobulin domains as well as α -integrin and collagen domains, which are required for cell‐cell interaction and immune system function in animals [2]. Thus, choanoflagellates are the simplest living system for studying the primary functions of proteins of multicellular organisms that are involved in cell-cell interaction and signal transduction. In this study, we cloned cDNA and determined the complete amino acid sequence of the MB7TM2 receptor from M. brevicollis —a homologue of the CIRL receptor, an adhesion GPS-containing G protein-coupled secretin-type receptor that was discovered by us earlier. The mini-family of the G protein-coupled receptors CIRL comprises natural hybrids of proteins of two classes—signaling receptors and cell adhesion molecules. CIRL-1 is the acceptor of the presynaptic neurotoxin α -latrotoxin and is presumably involved in the regulation of neuronal exocytosis [3, 4]. Characteristic features of these G protein-coupled secretin-type receptors are a two-subunit structure, an extended extracellular N-terminal part containing cell adhesion modules, and the presence of seven transmembrane segments and a G protein-coupled receptor proteolysis site (GPS domain). In the GPS domain, the precursor molecule undergoes proteolysis to form two subunits of the receptor. Genomes of mammals, insects, and worms, contain genes for approximately 30 adhesion G protein-coupled secretin-type receptors, which contain the GPS domain [5]. Currently, endogenous agonists of adhesion G protein-coupled receptors are unknown. In view of this, studies of the structural‐functional characteristics of the simplest homologues of these receptors are of interest since they help to clarify the fundamental properties of adhesion G protein-coupled receptors. Complete sequencing of M. brevicollis genome allowed us to discover a fragment that is homologous to the GPS domain of the CIRL receptor. To clone the fulllength cDNA of the putative ortholog CIRL, we obtained total cDNA of M. brevicollis . The M. brevicollis strain ATCC 50154 (obtained from the American Type Culture Collection) was cultured at room temperature in ATCC medium 1525 supplemented with living bacteria Enterobacter aerogenes (strain ATCC 13048) to a density of 10 6 –10 7 cells/ml. Total RNA was isolated according to Chomczynski [6]; polyadenylated mRNA was purified on oligo-dT-agarose (Invitrogen, United States). cDNA was obtained by reverse transcription using the Moloney murine leukemia virus reverse transcriptase (M-MuLV RT; Fermentas, Lithuania) and the oligo- dT 18 primer.