Highlights d Proteogenomics characterization of 218 pediatric brain tumor samples of 7 histologies d Proteomic clusters reveal actionable biological features spanning histological boundaries d Proteomics reveal downstream effects of DNA alterations not evident in transcriptomics d Kinase activity analyses provide insights into pathway activities and druggable targets

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BACKGROUND AND PURPOSE:Chordoma and chondrosarcoma of the skull base are rare tumors with overlapping presentations and anatomic imaging features but different prognoses. We hypothesized that these tumors might be distinguished by using diffusionweighted MR imaging.

Heterotrimeric G proteins mediate physiological processes ranging from phototransduction to cell migration. In the accepted model of G protein signaling, G␣␤␥ heterotrimers physically dissociate after activation, liberating free G␣ subunits and G␤␥ dimers. This model is supported by evidence obtained in vitro with purified proteins, but its relevance in vivo has been questioned. Here, we show that at least some heterotrimeric G protein isoforms physically dissociate after activation in living cells. G␣ subunits extended with a transmembrane (TM) domain and cyan fluorescent protein (CFP) were immobilized in the plasma membrane by biotinylation and cross-linking with avidin. Immobile CFP-TM-G␣ greatly decreased the lateral mobility of intracellular G␤ 1␥2-YFP, indicating the formation of stable heterotrimers. A GTPase-deficient (constitutively active) mutant of CFP-TM-G␣ oA lost the ability to restrict G␤1␥2-YFP mobility, whereas GTPase-deficient mutants of CFP-TM-G␣ i3 and CFP-TM-G␣ s retained this ability. Activation of cognate G proteincoupled receptors partially relieved the constraint on G␤ 1␥2-YFP mobility induced by immobile CFP-TM-G␣ oA and CFP-TM-G␣i3 but had no effect on the constraint induced by CFP-TM-G␣ s. These results demonstrate the physical dissociation of heterotrimers containing G␣ oA and G␣i3 subunits in living cells, supporting the subunit dissociation model of G protein signaling for these subunits. However, these results are also consistent with the suggestion that G protein heterotrimers (e.g., G␣ s) may signal without physically dissociating.cross-linking ͉ fluorescence recovery after photobleaching ͉ G protein-coupled receptors H eterotrimeric G proteins are known to exist in their inactive state as stable complexes of G␣ subunits and G␤␥ dimers. G␣ subunits cycle between inactive (GDP-bound) and active (GTP-bound) states, and the lifetime of the active state is limited by GTP hydrolysis. Biochemical studies have shown that active G protein heterotrimers dissociate into G␣-GTP and G␤␥ subunits in vitro (1). However, it has been argued that G protein subunits may not dissociate under more physiological conditions (2-5), and recent resonance energy transfer (RET) studies have suggested that G protein activation in cells involves subunit rearrangement rather than dissociation (4, 5). Physical dissociation of G protein heterotrimers has not been shown to occur in living cells. To address this question we developed a method to detect protein association and dissociation (Fig. 1A). In this method, one protein of an interacting pair is immobilized in the plasma membrane by an extracellular cross-linking agent. A decrease in the lateral mobility of a second protein [measured by using fluorescence recovery after photobleaching (FRAP)] indicates a binding interaction between the two. The mobility of this second protein is restored only if the partners dissociate. Here, we use this method to show that some G protein subunits physically dissociate in living cells, whereas other heterotrimers appear t...