Maturation of rat brain is accompanied by differential expression of the long and short splice variants of G(s)alpha protein: identification of cytosolic forms of G(s)alpha

Distribution of the a subunit of the stimulatory G protein (G(s)alpha) was analyzed in membrane and cytosolic (supernatant 200 000 g) fractions from r at cortex, thalamus and hippocampus during the course of post-natal develop ment. In parallel, changes in beta -adrenoceptor density and adenylyl cycla se activity were determined. Long (G(s)alphaL) and short (G(s)alphaS) varia nts of G(s)alpha were assessed by immunoblotting using specific polyclonal antisera reacting with both G(s)alpha isoforms. Post-natal development was associated with an increase in the total amount of brain G(s)alpha. G(s)alp haL was the dominant isoform of G(s)alpha 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 G(s)alphaS also elevated dur ing post-natal development, but more pronounced changes were found in cytos olic G(s)alphaS. Although only a small amount of G(s)alphaS (much smaller t han G(s)alphaL) was detected among soluble proteins shortly after birth, G( s)alphaS prevailed over G(s)alphaL at PD90. The G(s)alphaL/G(s)alphaS ratio decreased, respectively, from 3.2 to 1.2 and from 5.0 to 1.5 in the membra ne fractions of cortex and hippocampus, but remained almost constant in tha lamus between PD1 and 90. More dramatic changes were found in the cytosolic fractions of all studied brain regions: the G(s)alphaL/G(s)alphaS ratio de creased 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 th e membrane-cytosol balance of G(s)alpha proteins alters dramatically during the course of brain development. Both G(s)alphaL and G(s)alphaS were expre ssed in a region- and age-specific manner, which suggests different roles i n the maturation of the brain tissue. A cyc(-) reconstitutive assay of cyto solic G(s)alpha indicated that only approximate to 20% of this protein was functional, compared with membrane-bound G(s)alpha, and its ability to reco nstitute adenylyl cyclase activity increased during the course of maturatio n. The number of beta -adrenoceptors increased sharply during early post-na tal development but only slightly in adulthood, and both GTP- and isoproter enol-stimulated adenylate cyclase activity reached peak values around PD12.