Use of a cAMP BRET sensor to characterize a novel regulation of cAMP by the sphingosine 1-phosphate/G13 pathway.
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.
Jiang
Lily I
LI
Department of Pharmacology, Alliance for Cellular Signaling, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9196, USA. lily.jiang@utsouthwestern.edu
Collins
Julie
J
Davis
Richard
R
Lin
Keng-Mean
KM
DeCamp
Dianne
D
Roach
Tamara
T
Hsueh
Robert
R
Rebres
Robert A
RA
Ross
Elliott M
EM
Taussig
Ronald
R
Fraser
Iain
I
Sternweis
Paul C
PC
D051702:Activating Transcription Factor 6; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D050976:Basic-Leucine Zipper Transcription Factors / Q000378:metabolism*; D015374:Biosensing Techniques; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D015936:Complement C5a / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D006801:Humans; D007155:Immunologic Factors / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D008246:Lysophospholipids / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D037342:Pertussis Toxin / Q000494:pharmacology; D011458:Prostaglandins E / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000494:pharmacology; D049349:Receptors, Lysosphingolipid / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism; D014530:Uridine Diphosphate / Q000494:pharmacology
C487807:ATF6B protein, human; D051702:Activating Transcription Factor 6; D000318:Adrenergic beta-Agonists; D050976:Basic-Leucine Zipper Transcription Factors; D007155:Immunologic Factors; D008246:Lysophospholipids; D011458:Prostaglandins E; D034741:RNA, Small Interfering; D049349:Receptors, Lysosphingolipid; C060506:sphingosine 1-phosphate; D014530:Uridine Diphosphate; D015936:Complement C5a; D000242:Cyclic AMP; D037342:Pertussis Toxin; D007545:Isoproterenol; D013110:Sphingosine