Dear Editor, Vasopressin type 2 receptor (V2R) belongs to the vasopressin (VP)/oxytocin (OT) receptor subfamily of G protein-coupled receptors (GPCRs), which comprises at least four closely related receptor subtypes: V1aR, V1bR, V2R, and OTR. 1 These receptors are activated by arginine vasopressin (AVP) and OT, two endogenous nine-amino acid neurohypophysial hormones, which are thought to mediate a biologically conserved role in social behavior and sexual reproduction. 2 V2R is mainly expressed in the renal collecting duct principal cells and mediates the antidiuretic action of AVP by accelerating water reabsorption, thereby playing a vital role in controlling water homeostasis. Moreover, numerous gain-of-function and loss-offunction mutations of V2R have been identified and are closely associated with human diseases, including nephrogenic syndrome of inappropriate diuresis (NSIAD) and X-linked congenital nephrogenic diabetes insipidus (NDI). 3 Thus, V2R has attracted intense interest as a drug target. However, due to a lack of structural information, how AVP recognizes and activates V2R remains elusive, which hampers the V2R-targeted drug design. Here, we determined a 2.6 Å resolution cryo-EM structure of the full-length, Gs-coupled human V2R bound to AVP (Fig. 1 a; Supplementary information, Table S1). The Gs protein was engineered based on mini-Gs that was used in the crystal structure determination of the Gs-coupled adenosine A2A receptor (A2AR) to stabilize the V2R–Gs protein complex (Supplementary information, Data S1). 4 The final structure of the AVP–V2R–Gs complex contains all residues of AVP (residues 1–9), the Gαs Ras-like domain, Gβγ subunits, Nb35, scFv16, and the V2R residues from T31 to L3398. 57(superscripts refer to Ballesteros–Weinstein numbering 5). The majority of amino acid side chains, including AVP, transmembrane domain (TMD), all flexible intracellular loops (ICLs) and extracellular loops (ECLs) except for ICL3 and G185–G188 in ECL2, were well resolved in the model, refined against the EM density map (Fig. 1 a; Supplementary information, Figs. S1–S3). The complex structure can provide detailed information on the binding interface between AVP and helix bundle of the receptor, as well as the receptor–Gs interface. AVP occupies an orthosteric binding pocket in the TMD bundle composed of all TM helices and ECLs (Fig. 1 b, c; Supplementary information, Table S2). The most notable conformational feature of AVP is the tocin ring formed by a disulfide bridge between the first and sixth cysteine residues, presenting a “spoon-like” conformation (Supplementary information, Fig. S4a, b). The cyclic spoon head inserts deeply into the TMD core, while the C-terminal spoon tail stretches toward the ECLs of the receptor. Cys1P of AVP and Q962. 61, K1163. 29, Q1193. 32 of V2R form a stabilizing H-bond network, consistent with the decreased potency of the receptor mutants with these residues mutated (Fig. 1 b; Supplementary information, Fig. S4c, d). The hydroxyl group of Tyr2P forms an H-bonds with the main chain oxygen of L3127. 40, whereas its main chain CO group forms another H-bond with Q1744. 60(Fig. 1 b; Supplementary information, Fig. S4c). Phe3P buries in a hydrophobic cleft constituted by M1203. 33, M1233. 36, Y2055. 38, V2065. 39, I2095. 42, F2876. 51, F2886. 52, and Q2916. 55 (Fig. 1 b; Supplementary information, Fig. S4c). Other polar contacts are seen between Gln4P and R2025. 35, Gln4P and the backbone and side chain oxygens of Q2916. 55, as well as Asn5P and the main chain of A194ECL2 (Fig. 1 b, c; Supplementary information, Fig. S4c). Arg8P forms salt …