Glutamatergic neurotransmission is controlled by presynaptic metabotropic glutamate receptors (mGluRs). A subdomain in the intracellular carboxyl-terminal tail of group III mGluRs binds calmodulin and heterotrimeric guanosine triphosphate-binding protein (G protein) betagamma subunits in a mutually exclusive manner. Mutations interfering with calmodulin binding and calmodulin antagonists inhibit G protein-mediated modulation of ionic currents by mGluR 7. Calmodulin antagonists also prevent inhibition of excitatory neurotransmission via presynaptic mGluRs. These results reveal a novel mechanism of presynaptic modulation in which Ca(2+)-calmodulin is required to release G protein betagamma subunits from the C-tail of group III mGluRs in order to mediate glutamatergic autoinhibition.

D 1 and D 2 receptors, the "classical" dopamine receptor subtypes, are abundantly expressed in the basal ganglia and are important targets in pharmacotherapy, yet the basis for their neuromodulatory effects is not well understood at the cellular level. The D 2 -dopamine receptor is found (as an autoreceptor) on presynaptic nerve terminals of nigrostriatal projections and, postsynaptically, on the medium spiny neuron, the predominant nerve cell of the neostriatum (2). The excitatory drive for the medium spiny neuron is provided by glutamatergic afferents which through NMDA receptors trigger Ca 2ϩ influx (3). Hence, neuronal signal transduction by dopamine receptors proceeds in the presence of oscillating intracellular Ca 2ϩ concentrations and there is reason to assume that the signaling mechanism is interrelated with the intracellular Ca 2ϩ level. Calmodulin (CaM), 1 a small acidic protein, can be considered the primary decoder of Ca 2ϩ information in the cell. CaM has a Ca 2ϩ affinity of 10 Ϫ6 M and thus acts as a switch when the concentration rises from a resting value of ϳ10 Ϫ7 M to 10Calmodulin can be activated by persistent elevation of intracellular Ca 2ϩ and by Ca 2ϩ oscillations, as they occur on repeated depolarizations of nerve cells (4). It has long been known that major effectors regulated by the D 2 -dopamine receptor can be regulated by Ca 2ϩ and that these effector molecules are enriched in striatal neurons. In these instances, increases in Ca 2ϩ levels elicit effects similar to D 2 receptor activation. For example, Ca 2ϩ reduces the intracellular cAMP levels by inhibiting adenylyl cyclase type V (and type VI) and by activating CaM-sensitive phosphodiesterases, which break down cAMP; both type V adenylyl cyclase (5, 6) and a 63-kDa isoform of phosphodiesterase (PDE1B1) are expressed in striatal neurons (7,8). Another example for the cross-talk between D 2 receptor signaling and Ca 2ϩ /CaM is the target protein DARPP-32, an inhibitor of protein phosphatase 1. DARPP-32 is dephosphorylated on D 2 -dopamine receptor activation and thus becomes active; this effect is strongly enhanced by Ca 2ϩ /CaM through activation of calcineurin (9). These examples suggest that the signal transduced by Ca 2ϩ /CaM and signaling initiated by the intracellular D 2 receptor overlap and may add to each other.We have found in the primary peptide sequence of the human D 2 -dopamine receptor a CaM-binding motif, which is located in the NH 2 terminus of the third cytoplasmic loop of the receptor. In the present work, we report that CaM can convey * This work was supported by Austrian Science Foundation Grants P13097 (to M. F.) and P14273 (to C. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.¶ To whom correspondence should be addressed. E-mail: christian. nanoff@univie.ac.at.

Ca2؉ /calmodulin (Ca 2؉ /CaM) and the ␤␥ subunits of heterotrimeric G-proteins (G␤␥) have recently been shown to interact in a mutually exclusive fashion with the intracellular C terminus of the presynaptic metabotropic glutamate receptor 7 (mGluR 7). Here, we further characterized the core CaM and G␤␥ binding sequences. In contrast to a previous report, we find that the CaM binding motif localized in the N-terminal region of the cytoplasmic tail domain of mGluR 7 is conserved in the related group III mGluRs 4A and 8 and allows these receptors to also bind Ca 2؉ /CaM. Mutational analysis of the Ca 2؉ /CaM binding motif is consistent with group III receptors containing a conventional CaM binding site formed by an amphipathic ␣-helix. Substitutions adjacent to the core CaM target sequence selectively prevent G␤␥ binding, suggesting that the CaM-dependent regulation of signal transduction involves determinants that overlap with but are different from those mediating G␤␥ recruitment. In addition, we present evidence that G␤␥ uses distinct nonoverlapping interfaces for interaction with the mGluR 7 C-terminal tail and the effector enzyme adenylyl cyclase II, respectively. Although G␤␥-mediated signaling is abolished in receptors lacking the core CaM binding sequence, ␣ subunit activation, as assayed by agonist-dependent GTP␥S binding, was not affected. This suggests that Ca 2؉ /CaM may alter the mode of group III mGluR signaling from mono-(␣) to bidirectional (␣ and ␤␥) activation of downstream effector cascades.Glutamate receptors mediate excitatory neurotransmission at most synapses in the central nervous system. Ionotropic glutamate receptors, such as AMPA, kainate, and NMDA receptors are involved in fast neurotransmission. In contrast, G protein-coupled metabotropic glutamate receptors (mGluRs)

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Sphingosine kinase has been recognized as an essential signaling molecule that mediates the intracellular conversion of sphingosine to sphingosine-1-phosphate. In mast cells, induction of sphingosine kinase and generation of sphingosine-1-phosphate have been linked to the initial rise in Ca 2؉ , released from internal stores, and to degranulation. These events either precede or are concomitant with the activation of phospholipase C-␥ and the generation of inositol trisphosphate. Here we show that sphingosine kinase type 1 (SPHK1) interacts directly with the tyrosine kinase Lyn and that this interaction leads to the recruitment of this lipid kinase to the high-affinity receptor for immunoglobulin E (FcRI). The interaction of SPHK1 with Lyn caused enhanced lipid and tyrosine kinase activity. After FcRI triggering, enhanced sphingosine kinase activity was associated with FcRI in sphingolipid-enriched rafts of mast cells. Bone marrow-derived mast cells from Lyn ؊/؊ mice, compared to syngeneic wild-type cells, were defective in the initial induction of SPHK1 activity, and the defect was overcome by retroviral Lyn expression. These findings position the activation of SPHK1 as an FcRI proximal event.In addition to the importance of sphingolipids in membrane structure, the sphingomyelin pathway with its lipid products ceramide, sphingosine (S), and S-1-phosphate (S1P) has been recognized to function in a variety of signaling events (3, 36). Many of the sphingolipids generated along this pathway are secreted and bind specific cellular receptors on a variety of cell types. On the other hand, they also function intracellularly as "second messenger molecules" similar to the various intermediates of phosphoglyceride metabolism (3, 36). Processes such as apoptosis, differentiation, and cell activation are directly regulated or fine-tuned by ceramide and its derivatives. However, in contrast to glycerolipids, the individual concentrations of various intracellular sphingolipids serve to balance cell activation and inactivation. This concept has been termed the sphingolipid rheostat (3). Specific examples include ceramide and S1P, whose relative intracellular concentration regulates apoptosis; S and S1P, which regulates immunoglobulin E-antigen (IgE-Ag) sensitivity in mast cells; and ceramide and ceramide-1-phosphate, which regulates the process of phagocytosis in macrophages (2,14,29,37,42,43). An essential cellular checkpoint in the sphingomyelin signaling pathway is S kinase (SPHK), as its product S1P is able to counteract ceramide as well as S-mediated effects in apoptosis and effector function regulation (e.g., inhibition of cytokine induction by S in mast cells). In humans, two different isoforms and several splice variants of SPHK were described and cloned (34). While activation of SPHK1 has been firmly established (e.g., after FcεRI and Fc␥RI signaling), its relationship to other known mast cell signaling molecules is still unexplored (24, 37). Recent data on Fc␥RI positioned SPHK downstream of phospholipase D, with a de...