Adhesion G protein-coupled receptor VLGR1/ADGRV1 regulates cell spreading and migration by mechanosensing at focal adhesions

Abstract

Summary VLGR1 (very large G protein-coupled receptor-1) is by far the largest adhesion G protein-coupled receptor in humans. Homozygous pathologic variants of VLGR1 cause hereditary deaf blindness in Usher syndrome 2C and haploinsufficiency of VLGR1 is associated with epilepsy. However, its molecular function remains elusive. Herein, we used affinity proteomics to identify many components of focal adhesions (FAs) in the VLGR1 interactome. VLGR1 is localized in FAs and assembles in FA protein complexes in situ . Depletion or loss of VLGR1 decreases the number and length of FAs in hTERT-RPE1 cells and in astrocytes of Vlgr1 mutant mice. VLGR1 depletion reduces cell spread and migration kinetics as well as the response to mechanical stretch characterizing VLGR1 as a metabotropic mechanosensor in FAs. Our data reveal a critical role of VLGR1 in the FA function and enlighten potential pathomechanisms in diseases related to VLGR1.

Isolation and culturing of primary mouse astrocytes for the analysis of focal adhesion dynamics

“…(2021) N/A Experimental models: organisms/strains Mouse: C57BL/6J (P0 age) The Jackson Laboratory Stock no: 000664 Mouse: Vlgr1 /del7TM (P0 age) Breed on a C57BL/6 background McMillan and White (2004) Mouse: Drum B (P0 age) Breed on a C57BL/6 background Potter et al. (2016) and Kusuluri et al. (2021) Recombinant DNA RFP-Paxillin Rudolf E. Leube, Rick Horwitz Rudolf E. Leube, Rick Horwitz Software and algorithms ImageJ software National Institutes of Health (NIH) http://imagej.nih.gov/ij/download.html Focal adhesion analysis server https://faas.bme.unc.edu Berginski and Gomez (2013) Other Stereomicroscope Leica Microsystems Leica WILD M3B Cold light source Leica Microsystems Schott KL750 Inverted microscope Nikon Instruments Inc. Nikon Eclipse Ti2-E Spinning disc unit Yokogawa Electric Corporation CSU-W1 Leica confocal microscope Leica Microsystems DM6000B Centrifuge Eppendorf Centrifuge 5430 R Medium-sized scissors A. Dumont & Fils Cat#T5074 Dumont #3 curved forceps A. Dumont & Fils Cat#T504 Dumont #4 standard tip forceps A. Dumont & Fils Cat#T505 Dumont #7 standard tip forceps …”

“…We recently showed that the adhesion G protein coupled receptor (GPCR) ADGRV1/VLGR1 (very large G protein coupled-receptor-1) is a component of FAs and mutations in VLGR1 cause defects in FA growth and maturation processes ( Kusuluri et al., 2021 ). In the latter study, we used isolated primary BL/6 wild-type astrocytes, Vlgr1 / del7TM mice lacking the seven transmembrane domains (7TM) and the intracellular domain (ICD) of Vlgr1 ( McMillan and White, 2004 ), and mutant Drum B mice in which only a small portion of the N-terminal fragment is present and the entire C-terminal fragment is missing ( Potter et al., 2016 ; Kusuluri et al., 2021 ). A significant decrease in the number and length of FAs was observed in both Vlgr1 mutant mice compared with primary BL/6 wild-type astrocytes ( Figure 10 ) ( Kusuluri et al., 2021 ).…”

“…In the latter study, we used isolated primary BL/6 wild-type astrocytes, Vlgr1 / del7TM mice lacking the seven transmembrane domains (7TM) and the intracellular domain (ICD) of Vlgr1 ( McMillan and White, 2004 ), and mutant Drum B mice in which only a small portion of the N-terminal fragment is present and the entire C-terminal fragment is missing ( Potter et al., 2016 ; Kusuluri et al., 2021 ). A significant decrease in the number and length of FAs was observed in both Vlgr1 mutant mice compared with primary BL/6 wild-type astrocytes ( Figure 10 ) ( Kusuluri et al., 2021 ). For the quantifications, we applied the ImageJ protocol described above in step 67 to 85.…”

“…In addition, we present an optimized transfection and manual quantification approach for focal adhesion analysis in fixed and living cells. For complete details on the use and execution of this protocol, please refer to Kusuluri et al. (2021) .…”

“…For complete details on the use and execution of this protocol, please refer to Kusuluri et al. (2021) .…”