Dual-agonist occupancy of orexin receptor 1 and cholecystokinin A receptor heterodimers decreases G-protein-dependent signaling and migration in the human colon cancer cell line HT-29

Abstract

The orexin (OX1R) and cholecystokinin A (CCK1R) receptors play opposing roles in the migration of the human colon cancer cell line HT-29, and may be involved in the pathogenesis and pathophysiology of cancer cell invasion and metastasis. OX1R and CCK1R belong to family A of the G-protein-coupled receptors (GPCRs), but the detailed mechanisms underlying their functions in solid tumor development remain unclear. In this study, we investigated whether these two receptors heterodimerize, and the results revealed novel signal transduction mechanisms. Bioluminescence and Förster resonance energy transfer, as well as proximity ligation assays, demonstrated that OX1R and CCK1R heterodimerize in HEK293 and HT-29 cells, and that peptides corresponding to transmembrane domain 5 of OX1R impaired heterodimer formation. Stimulation of OX1R and CCK1R heterodimers with both orexin-A and CCK decreased the activation of Gαq, Gαi2, Gα12, and Gα13 and the migration of HT-29 cells in comparison with stimulation with orexin-A or CCK alone, but did not alter GPCR interactions with β-arrestins. These results suggest that OX1R and CCK1R heterodimerization plays an anti-migratory role in human colon cancer cells.

Keywords: Bioluminescence resonance energy transfer (BRET); Cholecystokinin A receptor; Förster resonance energy transfer (FRET); G-protein–coupled receptor (GPCR); Heterodimerization; Migration; Orexin (hypocretin) receptor.