Effect of deletion of the major brain G-protein ? subunit (?o) on coordination of G-protein subunits and on adenylyl cyclase activity

Abstract

Heterotrimeric G-proteins, composed of alpha and betagamma subunits, transmit signals from cell-surface receptors to cellular effectors and ion channels. Cellular responses to receptor agonists depend on not only the type and amount of G-protein subunits expressed but also the ratio of alpha and betagamma subunits. Thus far, little is known about how the amounts of alpha and betagamma subunits are coordinated. Targeted disruption of the alpha(o) gene leads to loss of both isoforms of alpha(o), the most abundant alpha subunit in the brain. We demonstrate that loss of alpha(o) protein in the brain is accompanied by a reduction of beta protein to 32+/-2% (n = 4) of wild type. Sucrose density gradient experiments show that all of the betagamma remaining in the brains of alpha(o)-/- mice sediments as a heterotrimer (s20,w = 4.4 S, n = 2), with no detectable free alpha or betagamma subunits. Thus, the level of the remaining betagamma subunits matches that of the remaining alpha subunits. Protein levels of alpha subunits other than alpha(o) are unchanged, suggesting that they are controlled independently. Coordination of betagamma to alpha occurs posttranscriptionally because the mRNA level of the predominant beta1 subtype in the brains of alpha(o)-/- mice was unchanged. Adenylyl cyclase can be positively or negatively regulated by betagamma. Because the level of other alpha subunits is unchanged and alpha(o) itself has little or no effect on adenylyl cyclase, we could examine how a large change in the level of betagamma affects this enzyme. Surprisingly, we could not detect any difference in the adenylyl cyclase activity between brain membranes from wild-type and alpha(o)-/- mice. We propose that alpha(o) and its associated betagamma are sequestered in a distinct pool of membranes that does not contribute to the regulation of adenylyl cyclase.

Coordinate expression of the α and β subunits of heterotrimeric G proteins involves regulation of protein degradation in CHO cells

“…This conclusion agrees well with a previous report showing that stable increases in expression of GKi1, GKi2, GKi3 or GKq are always accompanied by an increase in GL protein levels in NIH3T3 ¢broblasts [18]. It is also compatible with reports that the abrogation of GKi2 expression, by knocking out the gene, decreased GL expression in embryonic murine ¢broblasts [8], and that the deletion of GKo decreased GL expression in the mouse brain [7].…”

“…The formation of an inactive heterotrimer helps to maintain a low noise level and enhances the interaction of GK with its receptors [5]. Thus, the presence of GK in excess of GLQ or that of GLQ in excess of GK would result in the continuous activation of e¡ectors even in the absence of the stimulating signals [7]. Therefore, cells need to have mechanisms for strict coordinate regulation of GK and GLQ protein expression to maintain cellular homeostasis, and this study provides evidence of such mechanisms.…”

“…It is not known why GL expression is di¡erently regulated following the overexpressing of the constitutively active forms of GKs and GKi2. Nevertheless, we speculate that a di¡erence of cell lines could probably be the cause, because di¡erent cell lines may have di¡erent compositions of G proteins and di¡erent regulatory mechanisms for coordinating the expression of GK and GLQ proteins [7].…”

“…Similarly, the expression of GL mRNA remained unchanged, but its protein concentration decreased to about one-third in GKo3/3 mouse brain. This discrepancy between the expressions of mRNA and protein was also attributed to rapid protein deg- radation, though the increase in the degradation rate of GLQ was not analyzed [7].…”

“…The presence of GK in excess of GLQ or that of GLQ in excess of GK would result in the continuous activation of e¡ectors even in the absence of the stimulating signals [7]. Therefore, a cell needs to maintain a steady GLQ/GK ratio, which requires a regulatory mechanism to coordinate the expressions of GK and GLQ.…”