RGD-Binding Integrins Regulate MOR Distribution Into Functional Plasma Membrane Microdomains

Objectives: Opioid drugs are widely used in treating orofacial pain. Understanding molecular mechanisms of opioid receptor sensitivity will help develop new therapies. Integrins modulate opioid receptors affinity. When integrins bind ligands, they form functional microdomains (lipid rafts) and initiate signal transduction. Our aims are to show that integrins influence mu opioid receptor (MOR) signaling by regulating their spatial distribution; examine effects of different integrin ligands; evaluate male/female differences on MOR recruitment to rafts; determine if an intact membrane is necessary for these effects. Methods: Rat trigeminal ganglia cell cultures were treated with soluble or bead-bound integrin binding peptide RGD, inactive reverse peptide DGR or integrin beta-1-blocking antibody. Some cultures were treated with methyl-beta-cyclodextrin (MBCD) to remove plasma membrane cholesterol prior to treatment. Lipid rafts were isolated in discontinuous sucrose gradients. Fractions (0.5ml) were collected, concentrated and analyzed by SDS-PAGE/Western blot. MOR was detected using ECL. Results: Lipid raft fractions (#3-5) from soluble RGD and beta-1-blocking antibody treated male cultures contained 60 and 55% more MOR, respectively, than DGR treated cells. Female cultures treated with bead-bound RGD recruited 62% more MOR to lipid rafts than male cultures. The amount of MOR in lipid rafts after treatment with bead-bound RGD was 5.7 times higher in normal compared to cholesterol-depleted cultures. Conclusions: RGD recruits more MOR than beta-1-blockig antibody into lipid rafts. Hence, beta-1 is not the only integrin involved in MOR recruitment. Gender had a significant effect on the amount of MOR recruitment by integrins after ligand treatment. Cholesterol-depletion reduced MOR recruitment indicating that an intact plasma membrane is necessary. Acknowledgements: This work was supported by NIDCR, Grant #DE14318 for the CO STAR Program and NIDCR Grant #P01DA016719.