Coupling of intracellular Ca2؉ to cAMP increases may be important for some forms of synaptic plasticity. The type I adenylyl cyclase (I-AC) is a neural-specific, Ca 2؉ -stimulated enzyme that couples intracellular Ca 2؉ to cAMP increases. Since optimal cAMP levels may be crucial for some types of synaptic plasticity, mechanisms for inhibition of Ca 2؉ -stimulated adenylyl cyclases may also be important for neuroplasticity. Here we report that Ca 2؉ stimulation of I-AC is inhibited by activation of G i -coupled somatostatin and dopamine D 2 L receptors. This inhibition is due primarily to G i␣ and not ␤␥ subunits since coexpression of ␤␥-binding proteins with I-AC did not affect somatostatin inhibition. However, ␤␥ released from G s did inhibit I-AC, indicating that the enzyme can be inhibited by ␤␥ in vivo. Interestingly, type VIII adenylyl cyclase (VIII-AC), another Ca 2؉ -stimulated adenylyl cyclase, was not inhibited by G i -coupled receptors. These data indicate that I-AC and VIII-AC are differentially regulated by G i -coupled receptors and provide distinct mechanisms for interactions between the Ca 2؉ and cAMP signal transduction systems. We propose that I-AC may be particularly important for synaptic plasticity that depends upon rapid and transient cAMP increases, whereas VIII-AC may contribute to transcriptional-dependent synaptic plasticity that is dependent upon prolonged, Ca 2؉ -stimulated cAMP increases.

The type I Ca(2+)-sensitive adenylyl cyclase has been implicated in several forms of synaptic plasticity in vertebrates. Mutant mice in which this enzyme was inactivated by targeted mutagenesis show deficient spatial memory and altered long term potentiation (Wu, Z. L., Thomas, S. A., Villacres, E. C., Xia, Z., Simmons, M. L., Chavkin, C., Palmiter, R. D., and Storm, D. R. (1995) Proc. Natl Acad Sci. U. S. A. 92, 220-224). Long term potentiation in the CA1 region of the rat hippocampus develops during the first 2 weeks after birth and reaches maximal expression at postnatal day 15 with a gradual decline at later stages of development. Here we report that Ca(2+)-stimulated adenylyl cyclase activity in rat hippocampus, cerebellum, and cortex increases significantly between postnatal days 1-16. This increase appears to be due to enhanced expression of type I adenylyl cyclase rather than type VIII adenylyl cyclase, the other adenylyl cyclase that is directly stimulated by Ca2+ and calmodulin. Type I adenylyl cyclase mRNA in the hippocampus increased 7-fold during this developmental period. The developmental expression of Ca(2+)-stimulated adenylyl cyclase activity in mouse brain was attenuated in mutant mice lacking type I adenylyl cyclase. Changes in expression of the type I adenylyl cyclase during the period of long term potentiation development are consistent with the hypothesis that this enzyme is important for neuroplasticity and spatial memory in vertebrates.

We used a bacterially expressed fusion protein containing the entire cytoplasmic domain of the human leukemia inhibitory factor (LIF) receptor to study its phosphorylation in response to LIF stimulation. The dose-and time-dependent relationships for phosphorylation of this construct in extracts of LIF-stimulated 3T3-L1 cells were superimposable with those for the stimulation of mitogen-activated protein kinase (MAPK). Indeed, phosphorylation of the cytoplasmic domain of the low-affinity LIF receptor a-subunit (LIFR) in Mono Q-fractionated, LIF-stimulated 3T3-L1 extracts occurred only in those fractions containing activated MAPK; Ser-1044 served as the major phosphorylation site in the human LIFR for MAPK both in agonist-stimulated 3T3-L1 lysates and by recombinant extracellular signal-regulated kinase 2 in vitro. Expression in rat H-35 hepatoma cells of LIFR or chimeric granulocyte-colony-stimulating factor receptor (G-CSFR)-LIFR mutants lacking Ser-1044 failed to affect cytokinestimulated expression of a reporter gene under the control of the ,8-fibrinogen gene promoter but eliminated the insulin-induced attenuation of cytokine-stimulated gene expression. Thus, our results identify the human LIFR as a substrate for MAPK and suggest a mechanism ofheterologous receptor regulation ofLIFR signaling occurring at Ser-1044.Leukemia inhibitory factor (LIF) is a member of the family of multifunctional cytokines capable of stimulating numerous physiological processes in a variety of cells (1, 2). LIF, along with ciliary neurotrophic factor, interleukins (ILs) 6 and 11, and oncostatin M, constitutes a distinct subgroup of the cytokine family (3) in which each member has its own unique a-receptor subunit that associates with the shared receptor subunit gp130 to initiate transmembrane signaling (4-12).Early studies of LIF signaling have indicated activation of a pathway(s) involving both Tyr and Ser/Thr protein kinases (12, 13). Both the low-affinity LIF receptor a subunit (LIFR) and gpl3O associate with and stimulate members of the Jak/Tyk family of nonreceptor protein-tyrosine kinases (14). We (15) and others (16,17) have shown recently that activation of LIFR by agonist rapidly stimulates several components of the mitogen-activated protein kinase (MAPK) cascade, including MAPK kinase, the MAPK isozymes extracellular signal-regulated kinases (ERKs) 1 and 2, and S6 protein kinase activities against both S6 peptide and 40S ribosomes. In the current study, we show that the human LIFR can be phosphorylated at Ser-1044 by activated MAPK in vitro and suggest that this residue mediates a pathway for regulation of LIFR signaling after stimulation of a heterologous receptor system.

The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) plays an important regulatory role in developing and adult nervous systems. With the exception of the 5-HT 3 receptor, all of the cloned serotonin receptors belong to the G protein-coupled receptor superfamily. Subtypes 5-HT 6 and 5-HT 7 couple to stimulation of adenylyl cyclases through G s and display high affinities for antipsychotic and antidepressant drugs. In the brain, mRNA for 5-HT 6 is found at high levels in the hippocampus, striatum, and nucleus accumbens. 5-HT 7 mRNA is most abundant in the hippocampus, neocortex, and hypothalamus. To better understand how serotonin might control cAMP levels in the brain, we coexpressed 5-HT 6 or 5-HT 7A receptors with specific isoforms of adenylyl cyclase in HEK 293 cells. The 5-HT 6 receptor functioned as a typical G s -coupled receptor in that it stimulated AC5, a G s -sensitive adenylyl cyclase, but not AC1 or AC8, calmodulin (CaM)-stimulated adenylyl cyclases that are not activated by G s -coupled receptors in vivo. Surprisingly, serotonin activation of 5-HT 7A stimulated AC1 and AC8 by increasing intracellular Ca 2؉ . 5-HT also increased intracellular Ca 2؉ in primary neuron cultures. These data define a novel mechanism for the regulation of intracellular cAMP by serotonin. Serotonin (5-hydroxytryptamine, 5-HT)1 is a ubiquitous neurotransmitter that elicits a variety of physiological effects peripherally and centrally (1-3). A growing family of plasma membrane receptors bind 5-HT and mediate its cellular effects (4). All of the 5-HT receptors except 5-HT 3 belong to the superfamily of G protein-coupled receptors. The recently cloned 5-HT 6 (5, 6) and 5-HT 7 (7-10) receptors activate adenylyl cyclase(s) through the heterotrimeric G protein G s , and expression of these receptors in cultured cells couples serotonin to increases in cAMP (6,8). 5-HT 6 and 5-HT 7 display high affinities for antipsychotic and antidepressant drugs including clozapine, amoxapine, and amitryptiline (5-8), suggesting a role for these receptors in affective function. In the brain, the 5-HT 6 receptor is most highly expressed in the hippocampus, nucleus accumbens, striatum, and limbic regions, and the 5-HT 7 receptor is found in the hypothalamus, hippocampus, and cortex. The distribution of these receptors in brain is consistent with the hypothesis that they play a role in mood and affect (8,(11)(12)(13). In addition, the expression of the 5-HT 7 receptor in the suprachiasmatic nucleus (SCN) and the ability of serotonin and cAMP to advance the mammalian circadian rhythm indicates that 5-HT 7 plays an important role in circadian physiology (10, 14, 15). 5-HT 7 receptors are also expressed in glial cells (16,17) and at lower levels in peripheral tissues including the spleen, intestine, and vascular smooth muscle (7-9, 18).Many psychotherapeutic agents modulate the cAMP signaling pathway (19) and perturbation of this signal transduction system may contribute to some affective disorders. To date, nine distinct cDNA clones for mammalian...

Specific forms of synaptic plasticity such as long-term potentiation (LTP) are modulated by or require increases in cAMP. The various adenylyl cyclase isoforms possess unique regulatory properties, and thus cAMP increases in a given cell type or tissue in response to converging signals are subject to the properties of the adenylyl cyclase isoforms expressed. In most tissues, adenylyl cyclase activity is stimulated by neurotransmitters or hormones via stimulatory G-protein (Gs)-coupled receptors and is inhibited via inhibitory G-protein (Gi)-linked receptors. However, in the hippocampus, stimulation of Gi-coupled receptors potentiates Gs-stimulated cAMP levels. This effect may be associated with the regulatory properties of adenylyl cyclase types 2 and 4 (AC2 and AC4), isoforms that are potentiated by the betagamma subunit of Gi in vitro. Although AC2 has been shown to be stimulated by betagamma in whole cells, reports describing the sensitivity of AC4 to betagamma in vivo have yet to emerge. Our results demonstrate that Gs-mediated stimulation of AC4 is potentiated by betagamma released from activated Gi-coupled receptors in intact human embryonic kidney (HEK) 293 cells. Furthermore, we show that the AC2 and AC4 proteins are expressed in the mouse hippocampal formation and that they colocalize with MAP2, a dendritic and/or postsynaptic marker. The presence of AC2 and AC4 in the hippocampus and the ability of each of these enzymes to detect coincident activation of Gs- and Gi-coupled receptors suggest that they may play a crucial role in certain forms of synaptic plasticity by coordinating such overlapping synaptic inputs.