In T cells, cAMP-dependent protein kinase (PKA) type I colocalizes with the T cell receptor–CD3 complex (TCR/CD3) and inhibits T cell function via a previously unknown proximal target. Here we examine the mechanism for this PKA-mediated immunomodulation. cAMP treatment of Jurkat and normal T cells reduces Lck-mediated tyrosine phosphorylation of the TCR/CD3 ζ chain after T cell activation, and decreases Lck activity. Phosphorylation of residue Y505 in Lck by COOH-terminal Src kinase (Csk), which negatively regulates Lck, is essential for the inhibitory effect of cAMP on ζ chain phosphorylation. PKA phosphorylates Csk at S364 in vitro and in vivo leading to a two- to fourfold increase in Csk activity that is necessary for cAMP-mediated inhibition of TCR-induced interleukin 2 secretion. Both PKA type I and Csk are targeted to lipid rafts where proximal T cell activation occurs, and phosphorylation of raft-associated Lck by Csk is increased in cells treated with forskolin. We propose a mechanism whereby PKA through activation of Csk intersects signaling by Src kinases and inhibits T cell activation.

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Serotonin (5-hydroxytryptamine, 5-HT) receptor pre-mRNA is alternatively spliced in human tissue to produce three splice variants, h5-HT7(a), h5-HT7(b) and h5-HT7(d), which differ only in their carboxyl terminal tails. Using membranes from transiently and stably transfected HEK293 cells expressing the three recombinant h5-HT7 splice variants we compared their pharmacological profiles and ability to activate adenylyl cyclase. Using PCR on cDNA derived from various human tissues, the 5-HT7(a) and 5-HT7(b) splice variants were detected in every tissue examined. The h5-HT7(d) splice variant was detected in 13 of 16 tissues examined, with predominant expression in the heart, small intestine, colon, ovary and testis. All three h5-HT7 splice variants displayed high affinity binding for [3H]5-HT (pKd=8.8-8.9) in the presence and absence of 100 microM GTP and had similar binding affinities for all 17 ligands evaluated. In HEK293 cells expressing similar, high levels of receptor (approximately 10,000 fmol/mg protein), 5-CT (5-carboxamidotryptamine), 5-MeOT (5-methoxytryptamine) and 5-HT were full agonists while 8-OH-DPAT ((2R)-(+)-8-hydroxy-2-(di-n-propylamino)tetralin) was a partial agonist with relative efficacy of approximately 0.8. Even at this high receptor level, EC50 values for stimulation of adenylyl cyclase were 10- to 50-fold higher than the Kd values, indicating a lack of spare receptors. No significant differences in coupling to adenylyl cyclase were observed between the three splice variants over a wide range of receptor expression levels. For antagonists, binding affinities determined by displacement of [3H]5-HT binding and by competitive inhibition of 5-HT-stimulated adenylyl cyclase activity were essentially identical amongst the splice variants. These studies indicate that the three human splice variants are pharmacologically indistinguishable and that modifications of the carboxyl tail do not influence coupling to adenylyl cyclase.

1 Using membranes from stably or transiently transfected HEK293 cells cultured in 5-HT-free medium and expressing the recombinant human 5-HT 7 receptor splice variants (h5-HT 7(a) , h5-HT 7(b) and h5-HT 7(d) ), we compared their abilities to constitutively activate adenylyl cyclase (AC). 2 All h5-HT 7 splice variants elevated basal and forskolin-stimulated AC. The basal AC activity was reduced by the 5-HT 7 antagonist methiothepin and this e ect was blocked by mesulergine (neutral 5-HT 7 antagonist) indicating that the inhibitory e ect of methiothepin is inverse agonism at the 5-HT 7 receptor. 3 Receptor density correlated poorly with constitutive AC activity in stable clonal cell lines and transiently transfected cells. Mean constitutive AC activity as a percentage of forskolin-stimulated AC was signi®cantly higher for the h5-HT 7(b) splice variant compared to the h5-HT 7(a) and h5-HT 7(d) splice variants but only in stable cell lines. 4 All eight 5-HT antagonists tested inhibited constitutive AC activity of all splice variants in a concentration-dependent manner. No di erences in inverse agonist potencies (pIC 50 ) were observed between the splice variants. The rank order of potencies was in agreement and highly correlated with antagonist potencies (pK b ) determined by antagonism of 5-HT-stimulated AC activity (methiothepin4metergoline4mesulergine5clozapine5spiperone5ritanserin4methysergide4ketanserin).5 The e cacy of inverse agonism was not receptor level dependent and varied for several 5-HT antagonists between membrane preparations of transiently and stably transfected cells. 6 It is concluded that the h5-HT 7 splice variants display similar constitutive activity and inverse agonist properties.