Detailed neuronal distribution of GPR3 and its co-expression with EF-hand calcium-binding proteins in the mouse...

The G-protein coupled receptor 3 (GPR3), a member of the class A rhodopsin-type GPR family, constitutively activates Gαs proteins without any ligands. Although there have been several reports concerning the functions of GPR3 in neurons, the physiological roles of GPR3 have not been fully elucidated. To address this issue, we analyzed GPR3 distribution in detail using fluorescence-based X-gal staining in heterozygous GPR3 knockout/LacZ knock-in mice, and further investigated the types of GPR3-expressing neurons using fluorescent double labeling with various EF-hand Ca²⁺-binding proteins. In addition to the previously reported GPR3-expressing areas, we identified GPR3 expression in the basal ganglia and in many nuclei of the cranial nerves, in regions related to olfactory, auditory, emotional, and motor functions. In addition, GPR3 was not only observed in excitatory neurons in layer V of the cerebral cortex, the CA2 region of the hippocampus, and the lateral nucleus of the thalamus, but also in γ-aminobutyric acid (GABA)-ergic interneurons in the cortex, hippocampus, thalamus, striatum, and cerebellum. GPR3 was frequently co-expressed with neuronal Ca²⁺-binding protein 2 (NECAB2) in neurons in various regions of the central nervous system, especially in the hippocampal CA2, medial habenular nucleus, lateral thalamic nucleus, dorsolateral striatum, brainstem, and spinal cord anterior horn. Furthermore, GPR3 also co-localized with NECAB2 at the tips of neurites in differentiated PC12 cells. These results suggest that GPR3 and NECAB2 are highly co-expressed in specific neurons, and that GPR3 may modulate Ca²⁺ signaling by interacting with NECAB2 in specific areas of the central nervous system.