Selective targeting of ligand-dependent and -independent signaling by GPCR conformation-specific anti-US28 intrabodies
Timo W.M. De Groof, Nick D. Bergkamp, Raimond Heukers, Truc Giap, Maarten P. Bebelman, Richard Goeij-de Haas, Sander R. Piersma, Connie R. Jiménez, K. Christopher García, Hidde L. Ploegh, Marco Siderius, Martine J. Smit
Abstract
Abstract While various GPCRs, including US28, display constitutive, ligand-independent activity, it remains to be established whether ligand-dependent and -independent active conformations differ and can be selectively modulated. Previously, the agonist-bound conformation of US28 was stabilized and its structure was solved using the anti-US28 nanobody Nb7. Here we report the recognition of the constitutively active, apo-conformation of US28 by another nanobody VUN103. While the Nb7 intrabody selectively inhibits ligand-induced signaling, the VUN103 intrabody blocks constitutive signaling, indicating the existence of distinct US28 conformational states. By displacing Gα q protein, VUN103 prevents US28 signaling and reduces tumor spheroids growth. Overall, nanobodies specific for distinct GPCR conformational states, i.e. apo- and agonist-bound, can selectively target and discern functional consequences of ligand-dependent versus independent signaling.