Stachel-independent modulation of GPR56/ADGRG1 signaling by synthetic ligands directed to its extracellular region

Gabriel S. Salzmana,b,c, Shu Zhangc, Ankit Guptad, Akiko Koided,e, Shohei Koided,f,1, and Demet Araçc,g,1

aBiophysical Sciences Program, The University of Chicago, Chicago, IL 60637; bMedical Scientist Training Program, The University of Chicago, Chicago, IL 60637; cDepartment of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637; dPerlmutter Cancer Center, New York University Langone Medical Center, New York, NY 10016; eDepartment of Medicine, New York University School of Medicine, New York, NY 10016; fDepartment of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016; and gGrossman Institute for Neuroscience, Quantitative Biology and Human Behavior, The University of Chicago, Chicago, IL 60637

Edited by Robert J. Lefkowitz, Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC, and approved August 10, 2017 (received for review May 30, 2017) Adhesion G protein-coupled receptors (aGPCRs) play critical roles A mechanistic understanding of the biology mediated by aGPCRs, in diverse biological processes, including neurodevelopment and and their ECRs in particular, will be a critical milestone on the path cancer progression. aGPCRs are characterized by large and diverse to treating aGPCR-mediated pathologies. extracellular regions (ECRs) that are autoproteolytically cleaved The aGPCR ECRs are characterized by the presence of a from their membrane-embedded signaling domains. Although ECRs conserved juxtamembrane G protein-coupled receptor autopro- regulate receptor function, it is not clear whether ECRs play a direct teolysis-inducing (GAIN) domain (20) and various adhesion-type regulatory role in G-protein signaling or simply serve as a protective domains (located N-terminal to the GAIN domain), which allow cap for the activating “Stachel” sequence. Here, we present a mech- aGPCRs to bind protein ligands (5, 21). An autoproteolytic anistic analysis of ECR-mediated regulation of GPR56/ADGRG1, an aGPCR with two domains [pentraxin and laminin/neurexin/sex event occurs within the GAIN domain during aGPCR matura- tion in the endoplasmic reticulum, cleaving the receptor into

hormonebinding globulin-like (PLL) and G protein-coupled receptor BIOCHEMISTRY autoproteolysis-inducing (GAIN)] in its ECR. We generated a panel of two fragments: an N-terminal fragment (NTF; composed of the high-affinity monobodies directed to each of these domains, from N-terminal adhesion domains and the majority of the GAIN which we identified activators and inhibitors of GPR56-mediated sig- domain) and a C-terminal fragment (CTF; composed of the naling. Surprisingly, these synthetic ligands modulated signaling of a C-terminal β-strand of the GAIN domain termed “Stachel” or GPR56 mutant defective in autoproteolysis and hence, in Stachel pep- “stalk,” the 7TM, and the intracellular region) (Fig. 1). After tide exposure. These results provide compelling support for a ligand- cleavage, the NTF and CTF remain noncovalently but tightly induced and ECR-mediated mechanism that regulates aGPCR signaling associated throughout trafficking and localization to the plasma in a transient and reversible manner, which occurs in addition to the membrane (20, 22). The conservation of the GAIN domain Stachel-mediated activation. suggests that it plays a role in aGPCR function. In this article, the term ECR represents all N-terminal adhesion domains as adhesion GPCR | allostery | cell signaling | monobody | protein engineering Significance he G protein-coupled receptor (GPCR) superfamily exhibits Tgreat diversity with regard to the length and complexity of the extracellular region (ECR). Landmark mechanistic and func- G protein-coupled receptors enable cells to sense extracellular tional studies of GPCRs to date have almost exclusively focused signals and translate them into physiological responses. In addi- tion to a tra