GPR7 and GPR8 are two structurally related orphan G-protein coupled receptors, presenting high similarities with opioid and somatostatin receptors. Two peptides, L8 and L8C, derived from a larger precursor, were recently described as natural ligands for GPR8. L8 is a 23 amino acid peptide, while L8C is the same peptide with a C-terminus extension of 7 amino acids, running through a dibasic motif of proteolytic processing. Using as query the amino acid sequence of the L8 peptide, we have identified in DNA databases a human gene predicted to encode related peptides, and its mouse ortholog. By analogy with L8 and L8C, two peptides, named L7 and L7C could result from the processing of a 125 amino acid human precursor, through the alternative usage of a dibasic amino acid motif. The ability of L7, L7C, L8 and L8C to activate GPR7 and GPR8 was tested using an aequorin-based functional assay. In this assay, L7C was the most potent agonist of GPR7 (EC50= 50 +/- 11nM) followed by L7, L8 and L8C. L8 was the most potent agonist of GPR8 (EC50= 58 +/- 5nM) followed by L8C, L7C and L7. A C-terminally truncated form of human L7 (WYKPAAGHSSYSVGRAAGLL) was active on both GPR7 and GPR8, but with a lower potency than the L7 or L7C forms.
L8 and L7 peptides were iodinated, and binding experiments were performed on membranes obtained from CHO-K1 cells expressing GPR7 or GPR8. In competition binding experiments, L7C and L8C were both more potent than the shorter L7 and L8 peptides on GPR7, L7C displaying the highest affinity. In contrast, GPR8 displayed the highest affinity for L8, followed by L8C, L7 and L7C. To determine the natural coupling of the receptor to intracellular signalling pathways, CHO-K1 cell lines stably expressing GPR7 or GPR8, in the absence of exogenous transduction protein, were generated. Significant inhibition of forskolin-induced cAMP accumulation was observed for low concentrations of the four peptides in CHO-K1-GPR7 cells. Inhibition of cAMP accumulation was also observed in CHO-K1-GPR8 cells. The effect of L7 and L8 peptides on each receptor was strongly inhibited by pertussis toxin. No modification of phosphatidylinositol turnover was observed in COS-7 cells transiently expressing GPR7 or GPR8. The tissue distribution of the two peptide precursors, preproL7 (ppL7) and preproL8 (ppL8), was investigated by RT-PCR in peripheral tissues and central nervous system regions. Specific ppL7 transcripts were found at high levels in adult and fetal brain, substantia nigra, spinal cord, placenta and colorectal adenocarcinoma. Transcripts encoding the ppL8 precursors were detected at high levels by RT-PCR in the substantia nigra, lymphoblastic leukemia, fetal kidney, colorectal adenocarcinoma and trachea. Tissue distribution of the receptor transcripts was determined in parallel. Transcripts encoding GPR7 were detected at high levels in hippocampus, amygdala, trachea and lung carcinoma. GPR7 transcripts were also detected at moderate levels in fetal brain, pituitary gland and prostate. GPR8 transcripts were detected at high levels in caudate nucleus, hippocampus, amygdala, and at moderate levels in the adult brain, thalamus, parietal cortex, pituitary gland, adrenal gland, lymph nodes and lymphoblastic leukemia.
The ppL7 and ppL8 genes encode therefore the precursors of a class of peptide ligands, active on two receptor subtypes, GPR7 and GPR8. The mild preference of each receptor for specific peptide species, and the distinct tissue distribution of the receptor and peptide precursors suggest each ligand and receptor has partially overlapping but also specific roles in this signalling system.
Given the distribution of GPR7 and its ligands, this receptor constitute a candidate target for the treatment of various diseases and disorders, including appetite disorders and pain. The partnership will pursue this program by developing chemical agonists and antagonists. Partnering and licensing will be considered at a later stage of development of the program.