Latrophilins are essential for endothelial junctional fluid shear stress mechanotransduction

Abstract

Endothelial cell (EC) responses to fluid shear stress (FSS) are crucial for vascular development, adult physiology and disease. PECAM1 is an important transducer but earlier events remain poorly understood. We therefore investigated heterotrimeric G proteins in FSS sensing.Knockdown (KD) in ECs of single Gα proteins had little effect but combined depletion of Gαi and Gαq/11 blocked all known PECAM1-dependent responses. Re-expression of Gαi2 and Gαq but not Gαi1 and Gαi3 rescued these effects. Sequence alignment and mutational studies identified that K307 in Gαi2 and Gq/11 (Q306 in Gαi1/3), determines participation in flow signaling. We developed pull-down assays for measuring Gα activation and found that this residue, localized to the GPCR interface, determines activation by FSS. We developed a protocol for affinity purification of GPCRs on activated Gα's, which identified latrophilins (ADGRLs) as specific upstream interactors for Gαi2 and Gq/11. Depletion of latrophilin-2 blocked EC activation of Gαi2 and Gαq, downstream events in vitro, and flow-dependent vascular morphogenesis in zebrafish embryos. Surprisingly, latrophilin-2 depletion also blocked flow activation of two additional pathways activated at cell-cell junctions, Smad1/5 and Notch1, independently of Gα proteins.Latrophilins are thus central mediators of junctional shear stress mechanotransduction via Gα protein-dependent and -independent mechanisms.

Early events in endothelial flow sensing

“…One particularly puzzling aspect of the current data on GPCRs in flow signaling is their redundancy. Multiple, distinct GPCRs reportedly mediate the same events downstream of flow 115,136,138,140,146 . Some of this overlap may be due to erroneous results or interpretations.…”

Developmental Perspectives on Arterial Fate Specification

“…These findings fit well with the close proximity of all these players in cell-cell contacts. Recent data posted on BioRXIV report a requirement for the adhesion GPCR latrophilin-2, another junctional component (Tanaka et al, 2020). Latrophilin-2 is a GPCR studied mainly in neurons (Moreno-Salinas et al, 2019) but also expressed in ECs (Camillo et al, 2020;Tanaka et al, 2020).…”

“…Recent data posted on BioRXIV report a requirement for the adhesion GPCR latrophilin-2, another junctional component (Tanaka et al, 2020). Latrophilin-2 is a GPCR studied mainly in neurons (Moreno-Salinas et al, 2019) but also expressed in ECs (Camillo et al, 2020;Tanaka et al, 2020). This studied showed that it is required for flow activation of VEGFRs and downstream events via its GPCR function, and for flow activation of Notch signaling independent of G proteins (Tanaka et al, 2020).…”

“…Latrophilin-2 is a GPCR studied mainly in neurons (Moreno-Salinas et al, 2019) but also expressed in ECs (Camillo et al, 2020;Tanaka et al, 2020). This studied showed that it is required for flow activation of VEGFRs and downstream events via its GPCR function, and for flow activation of Notch signaling independent of G proteins (Tanaka et al, 2020). Importantly, latrophilin-2 is not required for EC responses to VEGF or Notch ligands in the absence of flow, indicating that it is critical for conferring flow sensitivity to these pathways (Tanaka et al, 2020).…”

“…This studied showed that it is required for flow activation of VEGFRs and downstream events via its GPCR function, and for flow activation of Notch signaling independent of G proteins (Tanaka et al, 2020). Importantly, latrophilin-2 is not required for EC responses to VEGF or Notch ligands in the absence of flow, indicating that it is critical for conferring flow sensitivity to these pathways (Tanaka et al, 2020). Despite these clues, a clear molecular mechanism is currently lacking.…”