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.

No abstract

Mel 1a melatonin receptors belong to the super-family of guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. So far, interest in Mel 1a receptor signaling has focused mainly on the modulation of the adenylyl cyclase pathway via pertussis toxin (PTX)-sensitive G proteins. To further investigate signaling of the human Mel 1a receptor, we have developed an antibody directed against the C terminus of this receptor. This antibody detected the Mel 1a receptor as a protein with an apparent molecular mass of approximately 60 kDa in immunoblots after separation by SDS-PAGE. It also specifically precipitated the 2-[125I]iodomelatonin (125I-Mel)-labeled receptor from Mel 1a-transfected HEK 293 cells. Coprecipitation experiments showed that G(i2), G(i3), and G(q/11) proteins couple to the Mel 1a receptor in an agonist-dependent and guanine nucleotide-sensitive manner. Coupling was selective since other G proteins present in HEK 293 cells, (G(i1), G(o), G(s), G(z), and G12) were not detected in receptor complexes. Coupling of the Mel 1a receptor to G(i) and G(q) was confirmed by inhibition of high-affinity 125I-Mel binding to receptors with subtype-selective G protein alpha-subunit antibodies. G(i2) and/or G(i3) mediated adenylyl cyclase inhibition while G(q/11) induced a transient elevation in cytosolic calcium concentrations in HEK 293 cells stably expressing Mel 1a receptors. Melatonin-induced cytosolic calcium mobilization via PTX-insensitive G proteins was confirmed in primary cultures of ovine pars tuberalis cells endogenously expressing Mel 1a receptors. In conclusion, we report the development of the first antibody recognizing the cloned human Mel 1a melatonin receptor protein. We show that Mel 1a receptors functionally couple to both PTX-sensitive and PTX-insensitive G proteins. The previously unknown signaling of Mel 1a receptors through G(q/11) widens the spectrum of potential targets for melatonin.

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.

Many membrane proteins incur a folding problem during biosynthesis; only a fraction thereof is exported from the endoplasmic reticulum (ER), because quality control is stringent. This is also true for G protein-coupled receptors. Here, we identify the deubiquitinating enzyme Usp4 as an interaction partner of the A 2a adenosine receptor, a G s -coupled receptor. Usp4 binds to the carboxyl terminus of the A 2A receptor and allows for its accumulation as deubiquinated protein. This relaxes ER quality control and enhances cell surface expression of functionally active receptor. The effect of Usp4 on the A 2A receptor was specific because 1) it was not seen in C-terminally truncated versions of the receptor; 2) it was not mimicked by Usp14, another member of the ubiquitin-specific protease family; and 3) it was not seen with the metabotropic glutamate receptor-5, another G protein-coupled receptor with a high propensity for intracellular retention. These observations show that deubiquinating enzymes can regulate quality control in the ER.