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Notes: Low-density membrane fragments (domains) were separated from the bulk of plasma membranes of 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 of trimeric G proteins and capacity of the DOP receptor to stimulate both the fusion protein-linked Gi1α and endogenous pertussis-toxin sensitive G proteins was measured as d-Ala2, d-Leu5-enkephalin stimulated high-affinity GTPase or guanosine-5′-[γ-35S]triphosphate ([35S]GTPγS) 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]GTPγS 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]GTPγS 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 × 10−8 and 3.2 ± 1.4 × 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 GTPγS 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.

Notes: Distribution of the α subunit of the stimulatory G protein (Gsα) was analyzed in membrane and cytosolic (supernatant 200 000 g) fractions from rat cortex, thalamus and hippocampus during the course of post-natal development. In parallel, changes in β-adrenoceptor density and adenylyl cyclase activity were determined. Long (GsαL) and short (GsαS) variants of Gsα were assessed by immunoblotting using specific polyclonal antisera reacting with both Gsα isoforms. Post-natal development was associated with an increase in the total amount of brain Gsα. GsαL was the dominant isoform of Gsα in the membrane fractions of all studied brain regions and its amount increased markedly between post-natal day (PD) 1 and 90. The level of membrane-bound GsαS also elevated during post-natal development, but more pronounced changes were found in cytosolic GsαS. Although only a small amount of GsαS (much smaller than GsαL) was detected among soluble proteins shortly after birth, GsαS prevailed over GsαL at PD90. The GsαL/GsαS ratio decreased, respectively, from 3.2 to 1.2 and from 5.0 to 1.5 in the membrane fractions of cortex and hippocampus, but remained almost constant in thalamus between PD1 and 90. More dramatic changes were found in the cytosolic fractions of all studied brain regions: the GsαL/GsαS ratio decreased sharply in cortex (from 14.1 to 0.9), hippocampus (from 3.7 to 0.8), and also in thalamus (from 9.5 to 0.5). These results demonstrate that the membrane–cytosol balance of Gsα proteins alters dramatically during the course of brain development. Both GsαL and GsαS were expressed in a region- and age-specific manner, which suggests different roles in the maturation of the brain tissue. A cyc− reconstitutive assay of cytosolic Gsα indicated that only ≈ 20% of this protein was functional, compared with membrane-bound Gsα, and its ability to reconstitute adenylyl cyclase activity increased during the course of maturation. The number of β-adrenoceptors increased sharply during early post-natal development but only slightly in adulthood, and both GTP- and isoproterenol-stimulated adenylate cyclase activity reached peak values around PD12.