title: delta-Opioid receptors exhibit high efficiency when activating trimeric G proteins in membrane domains.
abstract: Low-density membrane fragments (domains) were separated from the bulk of plasma membranes of human embryonic kidney (HEK)293 cells expressing a delta-opioid (DOP) receptor-Gi1alpha fusion protein by drastic homogenization and flotation on equilibrium sucrose density gradients. The functional activity of trimeric G proteins and capacity of the DOP receptor to stimulate both the fusion protein-linked Gi1alpha and endogenous pertussis-toxin sensitive G proteins was measured as d-Ala2, d-Leu5-enkephalin stimulated high-affinity GTPase or guanosine-5'-[gamma-35S]triphosphate ([35S]GTPgammaS) binding. The maximum d-Ala2-d-Leu5 enkephalin (DADLE)-stimulated GTPase was two times higher in low-density membrane fragments than in bulk of plasma membranes; 58 and 27 pmol/mg/min, respectively. The same difference was obtained for [35S]GTPgammaS binding. Contrarily, the low-density domains contained no more than half the DOP receptor binding sites (Bmax = 6.6 pmol/mg versus 13.6 pmol/mg). Thus, when corrected for expression levels of the receptor, low-density domains exhibited four times higher agonist-stimulated GTPase and [35S]GTPgammaS binding than the bulk plasma membranes. The regulator of G protein signaling RGS1, enhanced further the G protein functional activity but did not remove the difference between domain-bound and plasma membrane pools of G protein. The potency of the agonist in functional studies and the affinity of specific [3H]DADLE binding to the receptor were, however, the same in both types of membranes - EC50 = 4.5 +/- 0.1 x 10(-8) and 3.2 +/- 1.4 x 10(-8) m for GTPase; Kd = 1.2 +/- 0.1 and 1.3 +/- 0.1 nm for [3H]DADLE radioligand binding assay. Similar results were obtained when sodium bicarbonate was used for alkaline isolation of membrane domains. By contrast, detergent-insensitive membrane domains isolated following treatment of cells with Triton X100 exhibited no DADLE-stimulated GTPase or GTPgammaS binding. Functional coupling between the DOP receptor and cognate G proteins was also blocked by high-energy ultrasound and repeated freezing-thawing. Our data indicate, for the first time, that membrane domains isolated using 'detergent-free' procedures exhibit higher efficiency of coupling between a G protein-coupled receptor and its corresponding G protein(s) than bulk plasma membranes. Detergent-extraction diminishes these interactions, even when the receptor and G proteins are physically tethered together.
authors.affiliation: Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Lenka
authors.initials: L
authors.lastname: Bourova
authors.forename: Alexandra
authors.initials: A
authors.lastname: Kostrnova
authors.forename: Lucie
authors.initials: L
authors.lastname: Hejnova
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Moravcova
authors.forename: Hyo-Eun
authors.initials: HE
authors.lastname: Moon
authors.forename: Jiri
authors.initials: J
authors.lastname: Novotny
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
authors.forename: Petr
authors.initials: P
authors.lastname: Svoboda
mesh_terms: D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry*; D002499:Centrifugation, Density Gradient; D003902:Detergents / Q000737:chemistry / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D016308:Enkephalin, Leucine-2-Alanine / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000493:pharmacokinetics; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry*; D006801:Humans; D007668:Kidney / Q000737:chemistry / Q000166:cytology; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D017465:Receptors, Opioid, delta / Q000819:agonists / Q000737:chemistry*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D012995:Solubility / Q000187:drug effects; D013010:Sonication; D013347:Subcellular Fractions / Q000737:chemistry
chemical_list: D003902:Detergents; D017465:Receptors, Opioid, delta; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D016308:Enkephalin, Leucine-2-Alanine; D020962:Heterotrimeric GTP-Binding Proteins