Endocytosis is required for efficient mitogen-activated protein kinase (MAPK) activation by activated growth factor receptors. We examined if H-Ras and K-Ras proteins, which are distributed across different plasma membrane microdomains, have equal access to the endocytic compartment and whether this access is necessary for downstream signaling. Inhibition of endocytosis by dominant interfering dynamin-K44A blocked H-Ras but not K-Ras-mediated PC12 cell differentiation and selectively inhibited H-Ras-but not K-Rasmediated Raf-1 activation in BHK cells. H-Ras-but not K-Ras-mediated Raf-1 activation was also selectively dependent on phosphoinositide 3-kinase activity. Stimulation of endocytosis and endocytic recycling by wildtype Rab5 potentiated H-Ras-mediated Raf-1 activation. In contrast, Rab5-Q79L, which stimulates endocytosis but not endocytic recycling, redistributed activated H-Ras from the plasma membrane into enlarged endosomes and inhibited H-Ras-mediated Raf-1 activation. Rab5-Q79L expression did not cause the accumulation of wild-type H-Ras in enlarged endosomes. Expression of wild-type Rab5 or Rab5-Q79L increased the specific activity of K-Ras-activated Raf-1 but did not result in any redistribution of K-Ras from the plasma membrane to endosomes. These results show that H-Ras but not K-Ras signaling though the Raf/MEK/MAPK cascade requires endocytosis and endocytic recycling. The data also suggest a mechanism for returning Raf-1 to the cytosol after plasma membrane recruitment.

The small molecule AT₂R antagonist, EMA401, is in clinical development as a novel analgesic for the relief of neuropathic pain.

Augmented angiotensin II/AT₂R signaling in the DRGs of CCI rats is attenuated by EMA300 to block p38 MAPK and p44/p42 MAPK activation, a mechanism with clinical validity for alleviating neuropathic pain.

The mechanisms involved in angiotensin II type 1 receptor (AT 1 -R) trafficking and membrane localization are largely unknown. In this study, we examined the role of caveolin in these processes. Electron microscopy of plasma membrane sheets shows that the AT 1 -R is not concentrated in caveolae but is clustered in cholesterolindependent microdomains; upon activation, it partially redistributes to lipid rafts. Despite the lack of AT 1 -R in caveolae, AT 1 -R⅐caveolin complexes are readily detectable in cells co-expressing both proteins. This interaction requires an intact caveolin scaffolding domain because mutant caveolins that lack a functional caveolin scaffolding domain do not interact with AT 1 -R. Expression of an N-terminally truncated caveolin-3, CavDGV, that localizes to lipid bodies, or a point mutant, Cav3-P104L, that accumulates in the Golgi mislocalizes AT 1 -R to lipid bodies and Golgi, respectively. Mislocalization results in aberrant maturation and surface expression of AT 1 -R, effects that are not reversed by supplementing cells with cholesterol. Similarly mutation of aromatic residues in the caveolin-binding site abrogates AT 1 -R cell surface expression. In cells lacking caveolin-1 or caveolin-3, AT 1 -R does not traffic to the cell surface unless caveolin is ectopically expressed. This observation is recapitulated in caveolin-1 null mice that have a 55% reduction in renal AT 1 -R levels compared with controls. Taken together our results indicate that a direct interaction with caveolin is required to traffic the AT 1 -R through the exocytic pathway, but this does not result in AT 1 -R sequestration in caveolae. Caveolin therefore acts as a molecular chaperone rather than a plasma membrane scaffold for AT 1 -R.Lipid-based sorting mechanisms play an important role in the organization of the plasma membrane into microdomains (1-3). The biophysical properties of sphingolipids and cholesterol drive the spontaneous formation of lateral assemblies of liquid-ordered lipid rafts in a sea of liquid-disordered phospholipids. The biological importance of lipid rafts follows from the lateral segregation that they impose on membrane proteins. The differential distribution of plasma membrane proteins across raft and nonraft membranes in turn results in the concentration of specific groups of signaling proteins and lipids within discrete areas of the cell membrane (3-6). This increases the efficiency and specificity of signaling events by allowing more efficient interactions between proteins and by preventing cross-talk between different pathways.Caveolae are an abundant surface feature of many mammalian cells and represent a specific subtype of lipid raft. Functionally, caveolae have been implicated in endocytosis (7), potocytosis (8), transcytosis (9), apical transport (10), and cholesterol balance (11). Caveolae are identified by their characteristic morphology (flask-shaped, 55-65-nm diameter pits) and the presence of integral membrane proteins, termed caveolins, of which three mammalian isoforms have bee...