G protein-coupled receptors (GPCRs) comprise the largest family of membrane proteins in the human genome and mediate cellular responses to an extensive array of hormones, neurotransmitters, and sensory stimuli. While some crystal structures have been determined for GPCRs, most are for modified forms, showing little basal activity, and are bound to inverse agonists or antagonists1. Consequently, these structures correspond to receptors in their inactive states. The visual pigment rhodopsin is the only GPCR for which structures exist that are thought to be in the active state2,3. However, these structures are for the apoprotein or opsin form that does not contain the agonist all-trans retinal. We present here a crystal structure for the constitutively active rhodopsin mutant E113Q4-6 in complex with a peptide derived from the C-terminus of the G protein transducin (the GαCT peptide). Importantly, the protein appears to be in an active conformation, and retinal is retained in the binding pocket after photoactivation. Comparison with the structure of ground state rhodopsin7 suggests how translocation of the retinal β-ionone ring leads to a rotational tilt of transmembrane helix 6 (TM6), the critical conformational change upon activation8. A key feature of this conformational change is a reorganization of water mediated hydrogen-bonding networks between the retinal-binding pocket and three of the most conserved GPCR sequence motifs. For the first time we thus show how an agonist ligand can activate its GPCR.

The human cannabinoid receptor 1 (CB1) belongs to the G protein-coupled receptor (GPCR) family. Among the members of GPCR family, it has an exceptionally long extracellular Nterminal domain (N-tail) of 116 amino acids but has no typical signal sequence. This poses questions of how the long N-tail affects the biosynthesis of the receptor and of how it is inserted into the endoplasmic reticulum (ER) membrane. Here we have examined the process of membrane assembly of CB1 in the ER membrane and the maturation of the receptor from the ER to the plasma membrane. We find that the long N-tail cannot be efficiently translocated across the ER membrane, causing the rapid degradation of CB1 by proteasomes; this leads to a low level of expression of the receptor at the plasma membrane. The addition of a signal peptide at the N terminus of CB1 or shortening of the long N-tail greatly enhances the stability and cell surface expression of the receptor without affecting receptor binding to a cannabinoid ligand, CP-55,940. We propose that the N-tail translocation is a crucial early step in biosynthesis of the receptor and may play a role in regulating the stability and surface expression of CB1.

1 The ability of the endogenous fatty acid amide, cis-oleamide (ODA), to bind to and activate cannabinoid CB 1 and CB 2 receptors was investigated. The anatomical distribution of ODA-stimulated [ 35 S]GTPgS binding in rat brain sections was indistinguishable from that of HU210. Increases of similar magnitude were observed due to both agonists in the striatum, cortex, hippocampus and cerebellum. 7 ODA (10 mM) significantly inhibited forskolin-stimulated cyclic AMP (cAMP) accumulation in mouse neuroblastoma N1E 115 cells (P ¼ 0.02, n ¼ 11). ODA-mediated inhibition was completely reversed by 1 mM SR141716A (Po0.001, n ¼ 11) and was also reversed by pretreatment with 300 ng ml À1 pertussis toxin (Po0.001, n ¼ 6). 8 These data demonstrate that ODA is a full cannabinoid CB 1 receptor agonist. Therefore, in addition to allosteric modulation of other receptors and possible entourage effects due to fatty acid amide hydrolase inhibition, the effects of ODA may be mediated directly via the CB 1 receptor.

Human cannabinoid receptor 1 (CB 1 ) has attracted substantial interest as a potential therapeutic target for treating obesity and other obsessive disorders. An understanding of the mechanism governing the transition of the CB 1 receptor between its inactive and active states is critical for understanding how therapeutics can selectively regulate receptor activity. We have examined the importance of the Thr at position 210 in CB 1 in this transition, a residue predicted to be on the same face of the helix as the Arg of the DRY motif highly conserved in the G protein-coupled receptor superfamily. This Thr was substituted with Ile and Ala via mutagenesis, and the receptors, T210I and T210A, were expressed in HEK 293 cells. The T210I receptor exhibited enhanced agonist and diminished inverse agonist affinity relative to the wild type, consistent with a shift toward the active form. However, treatment with GTPγS to inhibit G protein coupling diminished the affinity change for the inverse agonist SR141716A. The decreased thermal stability of the T210I receptor and increased level of internalization of a T210I receptor-GFP chimera were also observed, consistent with constitutive activity. In contrast, the T210A receptor exhibited the opposite profile: diminished agonist and enhanced inverse agonist affinity. The T210A receptor was found to be more thermally stable than the wild type, and high levels of a T210A receptor-GFP chimera were localized to the cell surface as predicted for an inactive receptor form. These results suggest that T210 plays a key role in governing the transition between inactive and active CB 1 receptor states.Human cannabinoid receptor 1 (CB 1 ) 1 is a member of the G protein-coupled receptor (GPCR) superfamily and, as such, consists of seven α-helical membrane-spanning segments that mediate the effects of extracellular signaling molecules. As a consequence of ligand binding, rearrangements in these segments impact the association of the receptor with an intracellular G protein which, in turn, impacts biological activity. GPCRs are subdivided into five families based on sequence homology, and the cannabinoid receptors are classified as members of the family 1a rhodopsin-like receptors. The conformation of family 1a receptors is chararcterized, in part, by a salt bridge between the cytosolic ends of transmembane segment 3 (TM3), including the DRY motif, and transmembrane segment 6 (TM6). In the resting state, this ionic lock creates a kink in TM6 at the conserved CWXP motif (1,2). Upon activation, the salt bridge is disrupted concomitant with relaxation and rotation of TM6 relative to . Accompanying these molecular rearrangements is the exposure of a hydrophobic patch on the cytoplasmic surface of the receptor which may be key for G protein binding (6).Mutagenesis of the histamine receptor (7), the β 2 -adrenergic receptor (8), and the α 1B -adrenergic receptor (9) to remove functional groups involved in the ionic lock results in † This work was supported in part by National Institutes of...

Bispecific antibodies offer a promising approach for the treatment of cancer but can be challenging to engineer and manufacture. Here we report the development of PF-06671008, an extended-half-life dual-affinity re-targeting (DART ® ) bispecific molecule against P-cadherin and CD3 that demonstrates antibody-like properties. Using phage display, we identified anti-P-cadherin single chain Fv (scFv) that were subsequently affinity-optimized to picomolar affinity using stringent phage selection strategies, resulting in low picomolar potency in cytotoxic T lymphocyte (CTL) killing assays in the DART format. The crystal structure of this disulfide-constrained diabody shows that it forms a novel compact structure with the two antigen binding sites separated from each other by approximately 30 Å and facing approximately 90˝apart. We show here that introduction of the human Fc domain in PF-06671008 has produced a molecule with an extended half-life (~4.4 days in human FcRn knock-in mice), high stability (T m 1 > 68˝C), high expression (>1 g/L), and robust purification properties (highly pure heterodimer), all with minimal impact on potency. Finally, we demonstrate in vivo anti-tumor efficacy in a human colorectal/human peripheral blood mononuclear cell (PBMC) co-mix xenograft mouse model. These results suggest PF-06671008 is a promising new bispecific for the treatment of patients with solid tumors expressing P-cadherin.