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The adhesion class of G protein-coupled receptors (GPCRs) is the second largest family of GPCRs (33 members in humans). Adhesion GPCRs (aGPCRs) are defined by a large extracellular N-terminal region that is linked to a C-terminal seven transmembrane (7TM) domain via a GPCR-autoproteolysis inducing (GAIN) domain containing a GPCR proteolytic site (GPS). Most aGPCRs undergo autoproteolysis at the GPS motif, but the cleaved fragments stay closely associated, with the N-terminal fragment (NTF) bound to the 7TM of the C-terminal fragment (CTF). The NTFs of most aGPCRs contain domains known to be involved in cell-cell adhesion, while the CTFs are involved in classical G protein signaling, as well as other intracellular signaling. In this workshop report, we review the most recent findings on the biology, signaling mechanisms, and physiological functions of aGPCRs.

The precise and rapid construction of alleles through CRISPR/Cas9-mediated genome engineering renders Drosophila melanogaster a powerful animal system for molecular structure–function analyses and human disease models. Application of the ovoD co-selection method offers expedited generation and enrichment of scarlessly edited alleles without the need for linked transformation markers, which specifically in the case of exon editing can impact allele usability. However, we found that knockin procedures by homology-directed repair (HDR) under ovoD co-selection resulted in low transformation efficiency. This is likely due to repeated rounds of Cas9 cleavage of HDR donor and/or engineered genomic locus DNA, as noted for other CRISPR/Cas9 editing strategies before, impeding the recovery of correctly edited alleles. Here we provide a one-step protocol to improve the generation of scarless alleles by ovoD -co-selection with single-guide RNA (sgRNA) binding site masking. Using this workflow, we constructed human disease alleles for two Drosophila genes, unc-13/CG2999 and armadillo/CG11579. We show and quantify how a known countermeasure, the insertion of silent point mutations into protospacer adjacent motif (PAM) or sgRNA homology regions, can potently suppress unintended sequence modifications during CRISPR/Cas9 genome editing of D. melanogaster under ovoD co-selection. This strongly increased the recovery frequency of disease alleles. [ABSTRACT FROM AUTHOR]

In mammals, numerous organ systems are equipped with adhesion G protein-coupled receptors (aGPCRs) to shape cellular processes including migration, adhesion, polarity and guidance. All of these cell biological aspects are closely associated with tumor cell biology. Consistently, aberrant expression or malfunction of aGPCRs has been associated with dysplasia and tumorigenesis. Mounting evidence indicates that cancer cells comprise viscoelastic properties that are different from that of their non-tumorigenic counterparts, a feature that is believed to contribute to the increased motility and invasiveness of metastatic cancer cells. This is particularly interesting in light of the recent identification of the mechanosensitive facility of aGPCRs. aGPCRs are signified by large extracellular domains (ECDs) with adhesive properties, which promote the engagement with insoluble ligands. This configuration may enable reliable force transmission to the ECDs and may constitute a molecular switch, vital for mechano-dependent aGPCR signaling. The investigation of aGPCR function in mechanosensation is still in its infancy and has been largely restricted to physiological contexts. It remains to be elucidated if and how aGPCR function affects the mechanoregulation of tumor cells, how this may shape the mechanical signature and ultimately determines the pathological features of a cancer cell. This article aims to view known aGPCR functions from a biomechanical perspective and to delineate how this might impinge on the mechanobiology of cancer cells. [ABSTRACT FROM AUTHOR]