δ-Opioid receptors exhibit high efficiency when activating trimeric G proteins in membrane domains

摘要: Low-density membrane fragments (domains) were separated from the bulk of plasma membranes human embryonic kidney (HEK)293 cells expressing a δ-opioid (DOP) receptor-Gi1α fusion protein by drastic homogenization and flotation on equilibrium sucrose density gradients. The functional activity trimeric G proteins capacity DOP receptor to stimulate both protein-linked Gi1α endogenous pertussis-toxin sensitive was measured as d-Ala2, d-Leu5-enkephalin stimulated high-affinity GTPase or guanosine-5′-[γ-35S]triphosphate ([35S]GTPγS) binding. maximum d-Ala2-d-Leu5 enkephalin (DADLE)-stimulated two times higher in low-density than membranes; 58 27 pmol/mg/min, respectively. same difference obtained for [35S]GTPγS Contrarily, domains contained no more half binding sites (Bmax = 6.6 pmol/mg versus 13.6 pmol/mg). Thus, when corrected expression levels receptor, exhibited four agonist-stimulated membranes. regulator signaling RGS1, enhanced further but did not remove between domain-bound pools protein. potency agonist studies affinity specific [3H]DADLE were, however, types – EC50 = 4.5 ± 0.1 × 10−8 3.2 ± 1.4 × 10−8 m GTPase; Kd = 1.2 ± 0.1 1.3 ± 0.1 nm radioligand assay. Similar results sodium bicarbonate used alkaline isolation domains. By contrast, detergent-insensitive isolated following treatment with Triton X100 DADLE-stimulated GTPγS Functional coupling cognate also blocked high-energy ultrasound repeated freezing-thawing. Our data indicate, first time, that using ‘detergent-free’ procedures exhibit efficiency protein-coupled its corresponding protein(s) Detergent-extraction diminishes these interactions, even are physically tethered together.