Shear Stress–Dependent Downregulation of the Adhesion-G Protein–Coupled Receptor CD97 on Circulating Leukocytes upon Contact with Its Ligand CD55

Abstract

Adhesion G protein–coupled receptors (aGPCRs) are two-subunit molecules, consisting of an adhesive extracellular α subunit that couples noncovalently to a seven-transmembrane β subunit. The cooperation between the two subunits and the effect of endogenous ligands on the functioning of aGPCRs is poorly understood. In this study, we investigated the interaction between the pan-leukocyte aGPCR CD97 and its ligand CD55. We found that leukocytes from CD55-deficient mice express significantly increased levels of cell surface CD97 that normalized after transfer into wild-type mice because of contact with CD55 on both leukocytes and stromal cells. Downregulation of both CD97 subunits occurred within minutes after first contact with CD55 in vivo, which correlated with an increase in plasma levels of soluble CD97. In vitro, downregulation of CD97 on CD55-deficient leukocytes cocultured with wild-type blood cells was strictly dependent on shear stress. In vivo, CD55-mediated downregulation of CD97 required an intact circulation and was not observed on cells that lack contact with the blood stream, such as microglia. Notably, de novo ligation of CD97 did not activate signaling molecules constitutively engaged by CD97 in cancer cells, such as ERK and protein kinase B/Akt. We conclude that CD55 downregulates CD97 surface expression on circulating leukocytes by a process that requires physical forces, but based on current evidence does not induce receptor signaling. This regulation can restrict CD97–CD55-mediated cell adhesion to tissue sites.

Dihydromunduletone Is a Small-Molecule Selective Adhesion G Protein–Coupled Receptor Antagonist

“…We support a hypothesis where adhesion GPCR activation requires at least two separable events to occur in a prescribed order: 1) adhesion GPCR NTFs bind to anchored protein ligands, and 2) shear force generated by cell movement in relation to the anchored-ligand:NTF complex overcomes large energetic and entropic barriers that are required to dissociate the NTF from the CTF (Yona et al, 2008;Karpus et al, 2013;Langenhan et al, 2013;Scholz et al, 2015;Stoveken et al, 2015). NTF dissociation results in decryption of the adhesion GPCR tethered-peptide agonist, which rapidly and perhaps irreversibly binds its orthosteric site on the 7TM domain (CTF).…”

“…A current hypothesis which we favor that describes the dynamism of receptor activation consists of two components: 1) anchored protein ligands bind aGPCR N-terminal fragment (NTF) binding determinants, and 2) the action of shear force created by cell movement serves to dissociate the NTF from the CTF to release or decrypt the tethered agonist (Karpus et al, 2013;Langenhan et al, 2013;Scholz et al, 2015;Stoveken et al, 2015). We reconstituted aGPCRs, GPR56 (ADGRG1) and GPR110 (ADGRF1), with purified G protein heterotrimers and provided a biochemical demonstration that experimentally induced NTF dissociation dramatically enhanced aGPCRmediated G protein activation (Stoveken et al, 2015).…”

Gedunin- and Khivorin-Derivatives Are Small-Molecule Partial Agonists for Adhesion G Protein-Coupled Receptors GPR56/ADGRG1 and GPR114/ADGRG5

“…The final version may differ from this version. anchoring and physiological shear forces thought to induce holo-receptor dissociation leading to tethered-peptide-agonist exposure (Karpus et al, 2013;Stoveken et al, 2015).…”

“…We favor a hypothesis that both types of aGPCR ligands facilitate signaling by acting as anchors that affix aGPCR extracellular domains. Cell-mediated shear force then dissociates the two aGPCR fragments to release the tethered-peptideagonist from its concealed location so that it may engage its 7TM domain orthosteric binding site (Karpus et al, 2013;Langenhan et al, 2013;Scholz et al, 2015;Stoveken et al, 2015).…”

Adhesion G Protein–Coupled Receptors: From In Vitro Pharmacology to In Vivo Mechanisms

“…A constitutively active GPR56 mutant enhances interactions with b-arrestin 2 and ubiquitination of the receptor (Paavola et al, 2011). Further, ligand-induced downregulation has been demonstrated for CD97 in circulating leukocytes (Karpus et al, 2013). This downregulation of CD97 required shear stress and correlated with an increase in plasma levels of soluble CD97, suggesting that dissociation of the NTF triggers degradation of the CTF of the receptor.…”