Adhesion GPCRs (aGPCRs) are a subfamily of GPCRs that are involved in cell adhesion, cell proliferation, and cell migration in various tissues. G protein-coupled receptor proteolytic site (GPS) of aGPCR is required to cleave the extracellular domain autocatalytically, generating two fragments; a N-terminal fragment (NTF) and a C-terminal fragment (CTF) containing seven transmembrane structure. NTF can interact with CTF non-covalently after cleavage, however the physiological significance of the cleavage of aGPCR at GPS, and also the interaction between NTF and CTF have not been fully clarified yet. In this study, we first investigated the expression profiles of two aGPCRs, GPR56/ADGRG1, and LPHN1/ADGRL1 in mouse brain, and found that the NTF and CTF of GPR56 independently expressed in different brain region at different developmental stages. Immunoprecipitation of GPR56CTF co-immunoprecipitated LPHN1NTF from mouse brain and HEK293T cells expressing both fragments. Stimulation with LPHN1 ligand, α-Latrotoxin N4C (αLTXN4C), to cells expressing LPHN1NTF and GPR56CTF increased intracellular Ca2+ concentration ([Ca2+ ]i). We also demonstrated that GPR56KO mouse neurons attenuated their Ca2+ response to αLTXN4C. These results suggest the possibility of functional and chimeric complex containing LPHN1NTF and GPR56CTF in neuronal signal transduction.