What is the subunit of protein

Interaction partner of the alpha subunit of the stimulatory G protein, G (alpha) s

The alpha subunit of the stimulatory G protein, G (alpha) s, plays a central role in cellular signal transduction. G (alpha) s is a transmitter between a large number of G protein-coupled receptors (GPCR) and the effector adenylyl cyclase (AC) and stimulates the production of the secondary messenger substance cAMP. In contrast to other G proteins, apart from AC, only a few other effectors and no regulators of G (alpha) s activity are known. In the present work, therefore, new interaction partners of G (alpha) s were characterized via direct protein-protein interactions. For this purpose, two methods based on affinity purification were used, with which the bait protein G (alpha) s was isolated from mammalian cell lysates together with bound interaction partners. The isolated protein complexes were analyzed by SDS gel electrophoresis and subsequent mass spectrometry. For the functional characterization of the interaction of G (alpha) s with previously unknown interaction partners, recombinant G (alpha) s was used. However, in contrast to bovine G (alpha) s, murine orthologues could not be expressed and purified in sufficient quantities either in wild-type form or using C-terminal and internal His6 epitopes. For the functional characterization of the interaction partners, C-terminal His6-labeled bovine G (alpha) s was used. The mouse orthologue of a human regulator of G (alpha) s activity, human RGS-PX1, which was first described shortly before the start of the work, was examined in more detail. It was shown that the G (alpha) s activity is not influenced by either murine RGS-PX1 or human RGS-PX1, so that RGS-PX1 cannot be regarded as a regulator of G (alpha) s activity. In the search for new G (alpha) s interaction partners, 10 proteins interacting with inactive G (alpha) s and 29 with activated G (alpha) s were identified, including calmodulin, 14-3-3 (beta), 14-3 -3 (gamma) and 14-3-3 (zeta) as well as G (gamma) 10 and G (gamma) 12, whose interaction with G (alpha) s was characterized in more detail. An interaction of G (alpha) s with 14-3-3 proteins has not been confirmed. Calmodulin was confirmed as a G (alpha) s interaction partner by reciprocal co-purifications of isolated proteins. However, an influence of calmodulin on the G (alpha) s-catalyzed GTP hydrolysis could not be demonstrated. The interaction of G (alpha) s with G (gamma) 10 and G (gamma) 12 was shown for the first time in vitro in the present work by co-purification of the heterologously expressed proteins in insect cells and offers a valuable approach for future investigations into alternative, signal transduction of G (alpha) s that is not located in the plasma membrane.


The alpha subunit of the stimulatory G protein, G (alpha) s, plays a central role in cellular signal transduction. G (alpha) s acts as a transmitter between a number of G protein-coupled receptors (GPCRs) and the effector enzyme adenylyl cyclase (AC) thereby stimulating the production of the second messenger cAMP. As opposed to other G proteins, few effectors other than AC and no regulators of G (alpha) s activity are known to date. In the present study, direct protein-protein interactions of G (alpha) s and novel partners were characterized. Therefore, two methods based on affinity purification were used to isolate the bait protein G (alpha) s together with bound partners from mammalian cell lysates. The isolated protein complexes were analyzed using SDS-gel electrophoresis and mass spectrometry. For the functional characterization of its interaction with novel unknown partners in vitro, recombinant G (alpha) s was used. Unlike bovine G (alpha) s, neither wild type nor C-terminally or internally affinity tagged murine G (alpha) s orthologs could be recovered in sufficient amounts. Therefore, the functional characterization of novel interactors was performed using C-terminally His6 tagged bovine G (alpha) s. Recently, human RGS-PX1 was described as a novel regulator of G (alpha) s activity and therefore, the mouse ortholog was characterized in detail. In functional assays it was demonstrated that neither the mouse nor the human ortholog affects G (alpha) s activity, therefore RGS-PX1 cannot be regarded as a regulator of G (alpha) s activity. Using the affinity purification methods, 10 specific interactors for inactive G (alpha) s and 29 specific interactors for activated G (alpha) s were identified. Of these proteins, calmodulin, 14-3-3 (beta), 14-3-3 (gamma) and 14-3-3 (zeta) as well as G (gamma) 10 and G (gamma) 12 were further characterized with regard to their interaction with G (alpha) s. The interaction of G (alpha) s and the 14-3-3 isoforms was not confirmed. Calmodulin was confirmed as a G (alpha) s-interacting protein by means of reciprocal co-purification of the isolated proteins. A functional influence of calmodulin on G (alpha) s-catalysed GTP hydrolysis was not detected. In this study, the interaction of G (alpha) s with G (gamma) 10 and G (gamma) 12 was demonstrated for the first time by co-purification of these proteins from insect cells where they were heterologously expressed. This provides a valuable basis for future characterization of alternate, non-plasma membrane localized G (gamma) s signal transduction.