Abstract
G protein-coupled receptor kinase 5 (GRK5) deficiency has been linked recently to early Alzheimer disease (AD), but the mechanism by which GRK5 deficiency may contribute to AD pathogenesis remains elusive. Here we report that overexpression of dominant negative mutant of GRK5 (dnGRK5) in a cholinergic neuronal cell line led to decreased acetylcholine (ACh) release. This reduction was fully corrected by pertussis toxin, atropine (a nonselective muscarinic antagonist), or methoctramine (a selective M2/M4 muscarinic receptor antagonist). Consistent with results in cultured cells, high potassium-evoked ACh release in hippocampal slices from young GRK5 knock-out mice was significantly reduced compared with wild type littermates, and this reduced ACh release was also fully corrected by methoctramine. In addition, following treatment with the nonselective muscarinic agonist oxotremorine-M, M2, and M4 receptors underwent significantly reduced internalization in GRK5KO slices compared with wild type slices, as assessed by plasma membrane retention of receptor immunoreactivity, whereas M1 receptor internalization was not affected by loss of GRK5 expression. Moreover, Western blotting revealed no synaptic or cholinergic degenerative changes in young GRK5 knock-out mice. Altogether, these results suggest that GRK5 deficiency leads to a reduced hippocampal ACh release and cholinergic hypofunction by selective impairment of desensitization of presynaptic M2/M4 autoreceptors. Because this nonstructural cholinergic hypofunction precedes the hippocampal cholinergic hypofunction associated with structural cholinergic degeneration and cognitive decline in aged GRK5 knock-out mice, this nonstructural alteration may be an early event contributing to cholinergic degeneration in AD.
Highlights
G protein-coupled receptor kinase 5 (GRK5) deficiency has been linked recently to early Alzheimer disease (AD), but the mechanism by which GRK5 deficiency may contribute to AD pathogenesis remains elusive
In addition to Pertussis toxin (PTX), treatment with a nonselective muscarinic antagonist, atropine (1 M), and a selective M2/M4R antagonist, methoctramine (MT, 100 nM), each resulted in significantly increased ACh release and eliminated the differences among the three cell lines (Fig. 3, C and D). These results suggest that the functional difference of GRK5 in these three cell lines leads to altered ACh release, which is mediated by an altered function of PTX-sensitive G protein-coupled muscarinic M2 and/or M4 receptors
DnGRK5 was overexpressed to compete with the intrinsic GRK5, to create a functional GRK5 deficiency [16]. This functional GRK5 deficiency led to reduced ACh release, which could be corrected by preventing muscarinic receptor activation of Gi/Go proteins (PTX) and by antagonizing all muscarinic receptors or just M2/M4 receptors
Summary
The resultant PCR product containing the dnGRK5 and phrGFP-C mammalian expression vector (Stratagene, La Jolla, CA) were digested with BamHI/SalI and ligated to generate dnGRK5GFP fusion construct, with the humanized Renilla green fluorescent protein (hrGFP) tagged at C-terminal of the dnGRK5. The wtGRK5GFP and dnGRK5GFP fusions from the phrGFP-C vector were digested with BsiWI/AvrII and inserted into the MCS 1 of pVITRO1 (see Fig. 1A). The cells were cultured in Dulbecco’s modified Eagles’s medium supplied with 10% fetal bovine serum, 100 g/ml streptomycin, and 100 units/ml penicillin, as previously described [19]. The cells were incubated with medium containing [methyl-3H]choline chloride at a final concentration of 0.05 M (5 Ci/ml) for 2 h This low concentration of [3H]choline favors the selective uptake of choline through high affinity choline transporter (HACT) [20]. MGRK5 bGRK5 GAPDH a Sequence of bovine GRK5. b Sequence of hrGFP
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