Maturation of rat brain is accompanied by differential expression of the long and short splice variants of Gsα protein: Identification of cytosolic forms of Gsα

Abstract

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 postnatal 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. Postnatal 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 postnatal day (PD) 1 and 90. The level of membrane-bound GsαS also elevated during postnatal 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 postnatal development but only slightly in adulthood, and both GTP- and isoproterenol-stimulated adenylate cyclase activity reached peak values around PD12.