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.
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...
The endogenous opioid peptide endomorphin-1 (1) was modified by attachment of lactose to the N-terminus via a succinamic acid spacer to produce compound 2. The carbohydrate modification significantly improved the metabolic stability and membrane permeability of 2 while retaining μ-opioid receptor binding affinity and agonist activity. Analogue 2 produced dose-dependent antinociceptive activity following intravenous administration in a chronic constriction injury (CCI) rat model of neuropathic pain with an ED(50) of 8.3 (± 0.8) μmol/kg. The corresponding ED(50) for morphine was 2.6 (± 1.4) μmol/kg. Importantly, compound 2 produced dose-dependent pain relief after oral administration in CCI rats (ED(50) = 19.6 (± 1.2) μmol/kg), which was comparable with that of morphine (ED(50) = 20.7 (±3.6) μmol/kg). Antineuropathic effects of analogue 2 were significantly attenuated by pretreatment of animals with the opioid antagonist naloxone, confirming opioid receptor-mediated analgesia. In contrast to morphine, no significant constipation was produced by compound 2 after oral administration.
Background: The vanilloid receptor 1 (TRPV1) is critical in the development of inflammatory hyperalgesia. Several receptors including G-protein coupled prostaglandin receptors have been reported to functionally interact with the TRPV1 through a cAMP-dependent protein kinase A (PKA) pathway to potentiate TRPV1-mediated capsaicin responses. Such regulation may have significance in inflammatory pain. However, few functional receptor interactions that inhibit PKAmediated potentiation of TRPV1 responses have been described.
This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.
Specific point mutations in caveolin-3, a predominantly muscle-specific member of the caveolin family, have been implicated in limb-girdle muscular dystrophy and in rippling muscle disease. We examined the effect of these mutations on caveolin-3 localization and function. Using two independent assay systems, Raf activation in fibroblasts and neurite extension in PC12 cells, we show that one of the caveolin-3 point mutants, caveolin-3-C71W, specifically inhibits signaling by activated H-Ras but not by K-Ras. To gain insights into the effect of the mutant protein on H-Ras signaling, we examined the localization of the mutant proteins in fibroblastic cells and in differentiating myotubes. Unlike the previously characterized caveolin-3-DGV mutant, the inhibitory caveolin-3-C71W mutant reached the plasma membrane and colocalized with wild type caveolins. In BHK cells, caveolin-3-C71W associated with caveolae and in differentiating muscle cells with the developing T-tubule system. In contrast, the caveolin-3-P104L mutant accumulated in the Golgi complex and had no effect on H-Ras-mediated Raf activation. Inhibition by caveolin-3-C71W was rescued by cholesterol addition, suggesting that the mutant protein perturbs cholesterol-rich raft domains. Thus, we have demonstrated that a naturally occurring caveolin-3 mutation can inhibit signaling involving cholesterol-sensitive raft domains.
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