A two-state model for the diffusion of the A2A adenosine receptor in hippocampal neurons: agonist-induced switch to slow mobility is modified by synapse-associated protein 102 (SAP102).

The A2A receptor is a class A/rhodopsin-like G protein-coupled receptor. Coupling to its cognate protein, Gs, occurs via restricted collision coupling and is contingent on the presence of cholesterol. Agonist activation slows diffusion of the A2A adenosine receptor in the lipid bilayer. We explored the contribution of the hydrophobic core and of the extended C terminus by examining diffusion of quantum dot-labeled receptor variants in dissociated hippocampal neurons. Single particle tracking of the A2A receptor(1-311), which lacks the last 101 residues, revealed that agonist-induced confinement was abolished and that the agonist-induced decrease in diffusivity was reduced substantially. A fragment comprising the SH3 domain and the guanylate kinase domain of synapse-associated protein 102 (SAP102) was identified as a candidate interactor that bound to the A2A receptor C terminus. Complex formation between the A2A receptor and SAP102 was verified by coimmunoprecipitation and by tracking its impact on receptor diffusion. An analysis of all trajectories by a hidden Markov model was consistent with two diffusion states where agonist activation reduced the transition between the two states and, thus, promoted the accumulation of the A2A receptor in the compartment with slow mobility. Overexpression of SAP102 precluded the access of the A2A receptor to a compartment with restricted mobility. In contrast, a mutated A2A receptor (with (383)DVELL(387) replaced by RVRAA) was insensitive to the action of SAP102. These observations show that the hydrophobic core per se does not fully account for the agonist-promoted change in mobility of the A2A receptor. The extended carboxyl terminus allows for regulatory input by scaffolding molecules such as SAP102.

A single allele of Hdac2 but not Hdac1 is sufficient for normal mouse brain development in the absence of its paralog.

The histone deacetylases HDAC1 and HDAC2 are crucial regulators of chromatin structure and gene expression, thereby controlling important developmental processes. In the mouse brain, HDAC1 and HDAC2 exhibit different developmental stage- and lineage-specific expression patterns. To examine the individual contribution of these deacetylases during brain development, we deleted different combinations of Hdac1 and Hdac2 alleles in neural cells. Ablation of Hdac1 or Hdac2 by Nestin-Cre had no obvious consequences on brain development and architecture owing to compensation by the paralog. By contrast, combined deletion of Hdac1 and Hdac2 resulted in impaired chromatin structure, DNA damage, apoptosis and embryonic lethality. To dissect the individual roles of HDAC1 and HDAC2, we expressed single alleles of either Hdac1 or Hdac2 in the absence of the respective paralog in neural cells. The DNA-damage phenotype observed in double knockout brains was prevented by expression of a single allele of either Hdac1 or Hdac2. Strikingly, Hdac1(-/-)Hdac2(+/-) brains showed normal development and no obvious phenotype, whereas Hdac1(+/-)Hdac2(-/-) mice displayed impaired brain development and perinatal lethality. Hdac1(+/-)Hdac2(-/-) neural precursor cells showed reduced proliferation and premature differentiation mediated by overexpression of protein kinase C, delta, which is a direct target of HDAC2. Importantly, chemical inhibition or knockdown of protein kinase C delta was sufficient to rescue the phenotype of neural progenitor cells in vitro. Our data indicate that HDAC1 and HDAC2 have a common function in maintaining proper chromatin structures and show that HDAC2 has a unique role by controlling the fate of neural progenitors during normal brain development.

Reengineering the collision coupling and diffusion mode of the A2A-adenosine receptor: palmitoylation in helix 8 relieves confinement.

The A(2A)-adenosine receptor undergoes restricted collision coupling with its cognate G protein G(s) and lacks a palmitoylation site at the end of helix 8 in its intracellular C terminus. We explored the hypothesis that there was a causal link between the absence of a palmitoyl moiety and restricted collision coupling by introducing a palmitoylation site. The resulting mutant A(2A)-R309C receptor underwent palmitoylation as verified by both mass spectrometry and metabolic labeling. In contrast to the wild type A(2A) receptor, the concentration-response curve for agonist-induced cAMP accumulation was shifted to the left with increasing expression levels of A(2A)-R309C receptor, an observation consistent with collision coupling. Single particle tracking of quantum dot-labeled receptors confirmed that wild type and mutant A(2A) receptor differed in diffusivity and diffusion mode; agonist activation resulted in a decline in mean square displacement of both receptors, but the drop was substantially more pronounced for the wild type receptor. In addition, in the agonist-bound state, the wild type receptor was frequently subject to confinement events (estimated radius 110 nm). These were rarely seen with the palmitoylated A(2A)-R309C receptor, the preferred diffusion mode of which was a random walk in both the basal and the agonist-activated state. Taken together, the observations link restricted collision coupling to diffusion limits imposed by the absence of a palmitoyl moiety in the C terminus of the A(2A) receptor. The experiments allowed for visualizing local confinement of an agonist-activated G protein-coupled receptor in an area consistent with the dimensions of a lipid raft.

Essential role of B-Raf in oligodendrocyte maturation and myelination during postnatal central nervous system development.

Mutations in the extracellular signal-regulated kinase (ERK) pathway, particularly in the mitogen-activated protein kinase/ERK kinase (MEK) activator B-Raf, are associated with human tumorigenesis and genetic disorders. Hence, B-Raf is a prime target for molecule-based therapies, and understanding its essential biological functions is crucial for their success. B-Raf is expressed preferentially in cells of neuronal origin. Here, we show that in mice, conditional ablation of B-Raf in neuronal precursors leads to severe dysmyelination, defective oligodendrocyte differentiation, and reduced ERK activation in brain. Both B-Raf ablation and chemical inhibition of MEK impair oligodendrocyte differentiation in vitro. In glial cell cultures, we find B-Raf in a complex with MEK, Raf-1, and kinase suppressor of Ras. In B-Raf-deficient cells, more Raf-1 is recruited to MEK, yet MEK/ERK phosphorylation is impaired. These data define B-Raf as the rate-limiting MEK/ERK activator in oligodendrocyte differentiation and myelination and have implications for the design and use of Raf inhibitors.

Restricted collision coupling of the A2A receptor revisited: evidence for physical separation of two signaling cascades.

The A(2A)-adenosine receptor is a prototypical G(s) protein-coupled receptor but stimulates MAPK/ERK in a G(s)-independent way. The A(2A) receptor has long been known to undergo restricted collision coupling with G(s); the mechanistic basis for this mode of coupling has remained elusive. Here we visualized agonist-induced changes in mobility of the yellow fluorescent protein-tagged receptor by fluorescence recovery after photobleaching microscopy. Stimulation with a specific A(2A) receptor agonist did not affect receptor mobility. In contrast, stimulation with dopamine decreased the mobility of the D(2) receptor. When coexpressed in the same cell, the A(2A) receptor precluded the agonist-induced change in D(2) receptor mobility. Thus, the A(2A) receptor did not only undergo restricted collision coupling, but it also restricted the mobility of the D(2) receptor. Restricted mobility was not due to tethering to the actin cytoskeleton but was, in part, related to the cholesterol content of the membrane. Depletion of cholesterol increased receptor mobility but blunted activation of adenylyl cyclase, which was accounted for by impaired formation of the ternary complex of agonist, receptor, and G protein. These observations support the conclusion that the A(2A) receptor engages G(s) and thus signals to adenylyl cyclase in cholesterol-rich domains of the membrane. In contrast, stimulation of MAPK by the A(2A) receptor was not impaired. These findings are consistent with a model where the recruitment of these two pathways occurs in physically segregated membrane microdomains. Thus, the A(2A) receptor is the first example of a G protein-coupled receptor documented to select signaling pathways in a manner dependent on the lipid microenvironment of the membrane.

Heterotrimeric G protein-independent signaling of a G protein-coupled receptor. Direct binding of ARNO/cytohesin-2 to the carboxyl terminus of the A2A adenosine receptor is necessary for sustained activation of the ERK/MAP kinase pathway.

The A2A adenosine receptor is a prototypical G(s)-coupled receptor, but it also signals, e.g. to mitogen-activated protein (MAP) kinase, via a pathway that is independent of heterotrimeric G proteins. Truncation of the carboxyl terminus affects the strength of the signal through these alternative pathways. In a yeast two-hybrid interaction hunt, we screened a human brain library for proteins that bound to the juxtamembrane portion of the carboxyl terminus of the A2A receptor. This approach identified ARNO/cytohesin-2, a nucleotide exchange factor for the small (monomeric) G proteins of the Arf (ADP-ribosylation factor) family, as a potential interaction partner. We confirmed a direct interaction by mutual pull down (of fusion proteins expressed in bacteria) and by immunoprecipitation of the proteins expressed in mammalian cells. To circumvent the long term toxicity associated with overexpression of ARNO/cytohesin-2, we created stable cell lines that stably expressed the A2A receptor and where ARNO/cytohesin-2 or the dominant negative version E156K-ARNO/cytohesin-2 was inducible by mifepristone. Cyclic AMP accumulation induced by an A2A-specific agonist was neither altered by ARNO/cytohesin-2 nor by the dominant negative version. This was also true for agonist-induced desensitization. In contrast, expression of dominant negative E156K-ARNO/cytohesin-2 and of dominant negative T27N-Arf6 abrogated the sustained phase of MAP kinase stimulation induced by the A2A receptor. We therefore conclude that ARNO/cytohesin-2 is required to support the alternative, heterotrimeric G protein-independent, signaling pathway of A2A receptor, which is stimulation of MAP kinase.