The observed complexes are stable in that they are not disrupted by receptor activation or modulation of G protein ␣ subunit function. However, using a peptide that binds G␤␥ (␤ARKct), we show that G␤␥ is critical for dopamine receptor-Kir3 complex formation, but not for maintenance of the complex. We also provide evidence that Kir3 channels and another effector, adenylyl cyclase, are stably associated with the ␤ 2 -adrenergic receptor and can be co-immunoprecipitated by anti-receptor antibodies. Using bioluminescence resonance energy transfer, we have shown that in living cells under physiological conditions, ␤ 2 AR interacts directly with Kir3.1/3.4 and Kir3.1/3.2c heterotetramers as well as with adenylyl cyclase. All of these interactions are stable in the presence of receptor agonists, suggesting that these signaling complexes persist during signal transduction. In addition, we provide evidence that the receptor-effector complexes are also found in vivo. The observation that several G protein-coupled receptors form stable complexes with their effectors suggests that this arrangement might be a general feature of G protein-coupled signal transduction.

The ability of dopamine D(4) and D(2) receptors to activate extracellular signal-regulated kinases (ERKs) 1 and 2 was compared using Chinese hamster ovary (CHO-K1) cells transfected with D(4.2), D(4.4), D(4.7), and D(2L) receptors. Dopamine stimulation of D(4) or D(2L) receptors produced a transient, dose-dependent increase in ERK1/2 activity. Receptor-specific activation of the ERK mitogen-activated protein kinase (MAPK) pathway was confirmed using the D(2)-like receptor-selective agonist quinpirole, whereas the specific antagonist haloperidol blocked activation. MAPK stimulation was dependent on a pertussis-toxin-sensitive G protein (G(i/o)). trans-Activation of the platelet-derived growth factor (PDGF) receptor was an essential step in D(4) and D(2L) receptor-induced MAPK activation. PDGF receptor-selective tyrosine kinase inhibitors tyrphostin A9 and AG1295 abolished or significantly inhibited ERK1/2 activation by D(4) and D(2L) receptors. Dopamine stimulation of the D(4) receptor also produced a rapid increase in tyrosine phosphorylation of the PDGF receptor-beta. The Src-family tyrosine kinase inhibitor PP2 blocked MAPK activation by dopamine; however, this drug was also found to inhibit PDGF-BB-stimulated ERK activity and autophosphorylation of the PDGF receptor-beta. Downstream signaling pathways support the involvement of a receptor tyrosine kinase. The phosphoinositide 3-kinase inhibitors wortmannin and LY294002, protein kinase C inhibitors GF109203X and Calphostin C, dominant-negative RasN17, and the MEK inhibitor PD98059 significantly attenuated or abolished activation of MAPK by dopamine D(4) and D(2L) receptors. Our results indicate that D(4) and D(2L) receptors activate the ERK kinase cascade by first mobilizing signaling by the PDGF receptor, followed by the subsequent activation of ERK1/2 by pathways associated with this receptor tyrosine kinase.

No abstract

In Alzheimer's disease, accumulation of soluble oligomers of ␤-amyloid peptide is known to be highly toxic, causing disturbances in synaptic activity and neuronal death. Multiple studies relate these effects to increased oxidative stress and aberrant activity of calciumpermeable cation channels leading to calcium imbalance. The transient receptor potential melastatin 2 (TRPM2) channel, a Ca 2ϩ -permeable nonselective cation channel activated by oxidative stress, has been implicated in neurodegenerative diseases, and more recently in amyloid-induced toxicity. Here we show that the function of TRPM2 is augmented by treatment of cultured neurons with ␤-amyloid oligomers. Aged APP/PS1 Alzheimer's mouse model showed increased levels of endoplasmic reticulum stress markers, protein disulfide isomerase and phosphorylated eukaryotic initiation factor 2␣, as well as decreased levels of the presynaptic marker synaptophysin. Elimination of TRPM2 in APP/PS1 mice corrected these abnormal responses without affecting plaque burden. These effects of TRPM2 seem to be selective for ␤-amyloid toxicity, as ER stress responses to thapsigargin or tunicamycin in TRPM2 ؊/؊ neurons was identical to that of wild-type neurons. Moreover, reduced microglial activation was observed in TRPM2 ؊/؊ /APP/PS1 hippocampus compared with APP/PS1 mice. In addition, age-dependent spatial memory deficits in APP/PS1 mice were reversed in TRPM2 ؊/؊ /APP/PS1 mice. These results reveal the importance of TRPM2 for ␤-amyloid neuronal toxicity, suggesting that TRPM2 activity could be potentially targeted to improve outcomes in Alzheimer's disease.