Cyclic AMP-dependent protein kinase phosphorylates group III metabotropic glutamate receptors and inhibits their function as presynaptic receptors

The metabotropic glutamate receptors (mGluRs) belong to the class of GTP-binding protein (G-protein)-coupled receptors that contain seven transmembrane domains. Eight mGluR subtypes have been cloned and are classified into three groups. Group I mGluRs (mGluR1 and mGluR5) activate phospholipase C through coupling to the Gq class of G-proteins, and are selectively activated by 3,5-dihydroxyphenylglycine (DHPG). Group II (mGluR2 and 3) and group III (mGluR4, 6, 7, and 8) mGluRs inhibit adenylyl cyclase activity through coupling to the Gi/Go class of G-proteins, and are selectively activated by (2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine (DCG-IV) and (L)-2-amino-4-phosphonobutyric acid (L-AP4), respectively (for review, see Conn and Pin 1997). Splice variants have been found for both group I mGluRs and group III mGluRs. Rat mGluR1 exists in five splice forms. The mGluR1a subtype has a long intracellular C terminal tail whereas mGluR1b, mGluR1c, and mGluR1d have short C termini. The mGluR1e splice form is not a functional receptor but consists of only of the N-terminal domain of the protein (Pin et al. 1992; Tanabe et al. 1992; Pin and Duvoisin 1995; Mary et al. 1997). Similar to mGluR1a, splice variants of rat mGluR5 receptors (mGluR5a and 5b) possess a long intracellular C-terminal tail (Minakami et al. 1994). With the exception of mGluR6, each group III mGluR subtype, has two splice variants (mGluR4a and 4b, 7a and 7b, and 8a and 8b, respectively), each with a relatively short intracellular C-terminal tail (Thomsen et al. 1997; Corti et al. 1998). One primary function of group II and group III mGluRs in the CNS is to presynaptically reduce glutamate release and thereby inhibit excitatory transmission at glutamatergic synapses (for review, see Conn and Pin 1997). Increasing evidence suggests that the function of presynaptic mGluRs is tightly regulated by protein kinases. For instance, activation of protein kinase C (PKC) inhibits the function of multiple presynaptic group II and group III mGluR subtypes at several glutamatergic synapses (Swartz et al. 1993; Kamiya and Yamamoto 1997; Macek et al. 1998). Furthermore, cAMP-dependent protein kinase (PKA) inhibits the function of group II mGluRs at excitatory synapses in area CA3 of the hippocampus (Kamiya and Yamamoto 1997; Maccaferri et al. 1998) and at the medial perforant path-dentate gyrus (MPP-DG) synapse (Schaffhauser et al. 2000). We recently reported that PKA-induced inhibition of mGluR2 is mediated by direct phosphorylation of the receptor at a single site (Ser843) on its C-terminal tail. Phosphorylation of this site inhibits mGluR2-mediated responses by inhibiting coupling of the receptor to G-proteins (Schaffhauser et al. 2000). Whether PKA-induced phosphorylation of mGluRs is restricted to mGluR2 or is a general mechanism involved in the regulation of multiple presynaptic mGluRs is not known. Furthermore, it is not clear whether this response can only be obtained with adenylyl cyclase activators, such as forskolin, or whether it is physiologically relevant and can be elicited by agonists of receptors coupled to the activation of adenylyl cyclase (and PKA). We now report that PKA directly phosphorylates the C-terminal domain of several group III mGluR subtypes (mGluR4a, mGluR7a, and mGluR8a) as well as mGluR3. For all the mGluRs studied, PKA phosphorylates a single serine residue in their C-terminal region. In addition, the phosphorylation site is highly conserved in all group III mGluRs except for mGluR4b. Furthermore, activation of PKA inhibits the function of presynaptic mGluRs at multiple hippocampal synapses and this effect can be elicited by activation of β-adrenergic receptors when examining effects mediated by representative group III (mGluR7) and group II (mGluR2) mGluRs.