EP receptor activation of AC2 leads to cAMP production in non-raft and nuclear compartments of human ASMs, while β adrenoceptor signalling is broadly detected across microdomains. The activity of PDE4 appears to play a role in maintaining the integrity of compartmentalized EP receptor responses in these cells.

b 2 -Adrenoceptors (b 2 ARs) are concentrated in caveolar lipid raft domains of the plasma membrane in airway smooth-muscle (ASM) cells, along with adenylyl cyclase type 6 (AC6). This is believed to contribute to how these receptors can selectively regulate certain types of cAMP-dependent responses in these cells. The goal of the present study was to test the hypothesis that b 2 AR production of cAMP is localized to specific subcellular compartments using fluorescence resonance energy transfer-based cAMP biosensors targeted to different microdomains in human ASM cells. Epac2-MyrPalm and Epac2-CAAX biosensors were used to measure responses associated with lipid raft and nonraft regions of the plasma membrane, respectively. Activation of b 2 ARs with isoproterenol produced cAMP responses that are most readily detected in lipid raft domains. Furthermore, overexpression of AC6 somewhat paradoxically inhibited b 2 AR production of cAMP in lipid raft domains without affecting b 2 AR responses detected in other subcellular locations or cAMP responses to EP 2 prostaglandin receptor activation, which were confined primarily to nonraft domains of the plasma membrane. The inhibitory effect of overexpressing AC6 was blocked by inhibition of phosphodiesterase type 4 (PDE4) activity with rolipram, inhibition of protein kinase A (PKA) activity with H89, and inhibition of A kinase anchoring protein (AKAP) interactions with the peptide inhibitor Ht31. These results support the idea that overexpression of AC6 leads to enhanced feedback activation of PDE4 via phosphorylation by PKA that is part of an AKAP-dependent signaling complex. This provides insight into the molecular basis for localized regulation of cAMP signaling in human ASM cells.

Epidermal growth factor receptor (EGFR) plays roles in cell proliferation, inhibition of apoptosis and angiogenesis among others. It has long been thought that binding of the ligand induces the dimer formation of monomeric EGFR (the ''ligand-induced dimerization model''). Upon ligand binding, tyrosine residues in the C-terminal tail of EGFR are phosphorylated, and recruit effector proteins such as Shc1 and Grb2, which trigger activation of downstream signaling cascades. However, a pletora of recent studies indicate that EGFR exists in dimeric form (a preformed dimer), which is incompatible with the ligand-induced dimerization model. To understand the molecular mechanism of activation of EGFR, we directly observed interactions among EGFR, EGF labeled with two different fluorescent compounds, and GFP-tagged Shc1 by multi-color TIRF microscopy. Within 30 seconds of application of the dye-conjugated EGF to cultured cells expressing EGFR, three different fluorescent spots of EGF and Shc1 appeared on the cell surface. EGFR mutant that have no kinase activity could bind EGF but not recruit Shc1. Two different fluorescent colors derived from EGF molecules do not co-localize, and binding of a single EGF molecule induces Shc1 recruitment to EGFR. These results suggest that binding of a single EGF molecule can activate preformed EGFR dimer, consistently with negative cooperativity in EGF binding to EGFR.