Regulation of intracellular cyclic adenosine 3,5-monophosphate (cAMP) is integral in mediating cell growth, cell differentiation, and immune responses in hematopoietic cells. To facilitate studies of cAMP regulation we developed a BRET (bioluminescence resonance energy transfer) sensor for cAMP, CAMYEL (cAMP sensor using YFP-Epac-RLuc), which can quantitatively and rapidly monitor intracellular concentrations of cAMP in vivo. This sensor was used to characterize three distinct pathways for modulation of cAMP synthesis stimulated by presumed G s -dependent receptors for isoproterenol and prostaglandin E 2 . Whereas two ligands, uridine 5-diphosphate and complement C5a, appear to use known mechanisms for augmentation of cAMP via G q /calcium and G i , the action of sphingosine 1-phosphate (S1P) is novel. In these cells, S1P, a biologically active lysophospholipid, greatly enhances increases in intracellular cAMP triggered by the ligands for G s -coupled receptors while having only a minimal effect by itself. The enhancement of cAMP by S1P is resistant to pertussis toxin and independent of intracellular calcium. Studies with RNAi and chemical perturbations demonstrate that the effect of S1P is mediated by the S1P 2 receptor and the heterotrimeric G 13 protein. Thus in these macrophage cells, all four major classes of G proteins can regulate intracellular cAMP.Cyclic adenosine 3Ј,5Ј-monophosphate (cAMP), a ubiquitous second messenger, mediates a wide range of cellular functions including cell metabolism (1), cell proliferation and differentiation (1), immune responses (2, 3), memory formation (4), and cardiac contractility (5). Canonically, the concentration of intracellular cAMP is regulated by two distinct families of enzymes. The transmembrane adenylyl cyclases (ACs) 3 synthesize cAMP from adenosine triphosphate (6, 7), whereas the cAMP-specific phosphodiesterases metabolize cAMP to biologically inactive adenosine 5Ј-monophosphate (8, 9). ACs are primarily activated by G␣ s but their activities can also be differentially regulated by G␣ i , G␤␥, or Ca 2ϩ (10, 11). The activities of various phosphodiesterases can be regulated by protein kinase A (PKA), extracellular-regulated kinase (ERK), phosphoinositide 3-kinase, and the concentration of cAMP itself (12-16). Thus integration of signaling by stimuli that can regulate the intracellular concentration of cAMP will depend strongly on the various pathways and the subtypes of ACs and phosphodiesterases expressed in individual cells at any given time.Assessment of the regulation of intracellular cAMP in vivo has only become possible recently. Zaccolo et al. (17) first described a FRET sensor for cAMP based on the cAMP binding domain of PKA. Subsequently, several reports have described FRET sensors for cAMP based on binding of the nucleotide to the Epac proteins (18 -21). While these FRET sensors have been effective for measuring changes and localization of cAMP in single cells, measurements are tedious. Furthermore, the requirement for excitation of donor molecules pro...

RNAi is proving to be a powerful experimental tool for the functional annotation of mammalian genomes. The full potential of this technology will be realized through development of approaches permitting regulated manipulation of endogenous gene expression with coordinated reexpression of exogenous transgenes. We describe the development of a lentiviral vector platform, pSLIK (single lentivector for inducible knockdown), which permits tetracycline-regulated expression of microRNA-like short hairpin RNAs from a single viral infection of any naïve cell system. In mouse embryonic fibroblasts, the pSLIK platform was used to conditionally deplete the expression of the heterotrimeric G proteins G␣12 and G␣13 both singly and in combination, demonstrating the G␣13 dependence of serum response element-mediated transcription. In RAW264.7 macrophages, regulated knockdown of G␤2 correlated with a reduced Ca 2؉ response to C5a. Insertion of a GFP transgene upstream of the G␤2 microRNA-like short hairpin RNA allowed concomitant reexpression of a heterologous mRNA during tetracycline-dependent target gene knockdown, significantly enhancing the experimental applicability of the pSLIK system. G protein ͉ tetracycline

CD47 is a unique member of the Ig superfamily with a single extracellular Ig domain followed by a multiply membrane-spanning (MMS) domain with five transmembrane segments, implicated in both integrin-dependent and -independent signaling cascades. Essentially all functions of CD47 require both the Ig and MMS domains, raising the possibility that interaction between the two domains is required for normal function.

] i is independent of actin and rafts but is insufficient to explain CD47 cooperation with TCR in IL-2 synthesis. Full synergy with TCR requires CD47 localization to membrane rafts where ligation leads to TCR-independent signals causing actin polymerization and PKC translocation.