Akademik Veri Yönetim Sistemi
5. Uluslararası/34. Ulusal Biyofizik Kongresi, İzmir, Türkiye, 6 - 09 Eylül 2023, ss.231
Aim: G Protein-Coupled Receptor 37 (GPR37) is an orphan G protein-coupled receptor associated to Parkinson’s
disease neuropathology. Previous studies suggested that GPR37 is activated by prosaptide TX14(A), which is short
synthetic peptide derived from the neurotrophic sequence of prosaposin, and therefore, prosaptide TX14(A) is
proposed as endogenous ligand of GPR37. However, due to numerous discrepancies associated with pairing of
proposed ligand with the receptor, GPR37 remains an orphan receptor. Here, the aim of the study is to investigate
ligand-receptor binding using a single molecule technique called fluorescence correlation spectroscopy (FCS).
Materials and Methods: tGFP-tagged Human GPR37 plasmid and control plasmid pCMV6, mammalian vector
w/C terminal tGFP tag, were bought from Origene (Rockville, MD). NIH/3T3 (ATCC, Manassas, VA) cells were
maintained in a culture medium consisting of Dulbecco’s modified Eagle’s medium with 4500 mg glucose/L, 110
mg sodium pyruvate/L supplemented with 10% fetal bovine serum, glutamine (2 mM), and penicillin-streptomycin
(1%). For transient expression, NIH/3T3 cells were seeded in μ-Slide 8 well, #1.5 polymer coverslip (ibidi,
Gräfelfing, Germany,) at a density of 5× 104 cells per well. Transfections were performed by adding 100 ng of
GPR37 encoding plasmids per well using Lipofectamine 3000 (Invitrogen, Carlsbad, CA). Prosaptide TX14(A)
were purchased from Merck (Darmstadt, Germany). Twenty-four hours after transfection, transiently expressed
NIH/3T3 cells were treated with 0.1 μM-1 μM TX14(A) at 37°C for 40 min. Then the cells were subjected to FCS
measurements at a confocal Zeiss (Oberkochen, Germany) LSM 780 microscope.
Results: We observed changes in the diffusion coefficient of free and agonist bound GPR37 in NIH-3T3 cells.
Results demonstrate that the presence of the agonist modulates GPCR37's mobility and that agonist binding alters
the receptor's diffusion dynamics.
Conclusion: This study demonstrates GPR37 interaction with its agonist prosaptide TX14(A) at the plasma
membrane with fluorescence spectroscopy-based data.
Keywords: G Protein- Coupled Receptor 37; Fluorescence Correlation Spectroscopy; Diffusion; Agonist