Cloning and Expression of a cDNA for a Putative G Protein-Coupled Receptor from the Hepatopancreas of the Crayfish, Procambarus clarkii

Abstract

The authors cloned a novel cDNA encoding a putative G protein-coupled receptor (GPCR) from the hepatopancreas of the crayfish, Procambarus clarkii, using a screening approach with synthetic oligonucleotides. The oligonucleotides were designed homologous to the transmembrane spanning domain III of previously cloned receptors from various living organisms. Sequence analysis revealed that one of the positive clones contained a cDNA insertion of 3489 bp representing the mRNA coding for a part of a putative GPCR (termed HP1R). The clone was truncated at the 5′ end. The long 3′-untranslated regions (UTR) of 2446 bp contained three typical AATAAA censensus sequences for mRNA polyadenylation followed by the poly(A) tail at the 3′ end. To obtain a full-length cDNA clone, rapid amplification of the 5′ cDNA ends (5′ RACE) technique was then applied. Sequence analysis revealed that the full-length cDNA clone had an open-reading frame of 1116 bp with a 103-bp 5′-UTR. The predicted amino acid sequence of HP1R was 372 residues long. Hydropathicity analysis of HP1R suggested the presence of seven transmembrane domains. The database search revealed that the predicted sequence is most closely related to probable GPCR AH9.1 from Caenorhabditis elegans (27% identity, 48% homology). In addition, HP1R has lower homologies with receptors for somatostatin, opioid, dopamine, adrenalin, and so on. The similarity implied that HP1R is a new member of putative GPCRs whose endogenous ligands were unknown. In addition, RT–PCR analysis suggested that the transcript was expressed predominantly in the hepatopancreas but poorly in the muscle or brain.