Crystal Structure of the PAC1R Extracellular Domain Unifies a Consensus Fold for Hormone Recognition by Class B G-Protein Coupled Receptors

Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR). Crystal structures of a number of Class B GPCR extracellular domains (ECD) bound to their respective peptide hormones have revealed a consensus mechanism of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology of the ECD and a distinct mode of ligand recognition. Here we report a 1.9 Å crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors

Human Cannabinoid 1 GPCR C-Terminal Domain Interacts with Bilayer Phospholipids to Modulate the Structure of its Membrane Environment

G protein-coupled receptors (GPCRs) play critical physiological and therapeutic roles. The human cannabinoid 1 GPCR (hCB1) is a prime pharmacotherapeutic target for addiction and cardiometabolic disease. Our prior biophysical studies on the structural biology of a synthetic peptide representing the functionally significant hCB1 transmembrane helix 7 (TMH7) and its cytoplasmic extension, helix 8 (H8), [hCB1(TMH7/H8)] demonstrated that the helices are oriented virtually perpendicular to each other in membrane-mimetic environments. We identified several hCB1(TMH7/H8) structure-function determinants, including multiple electrostatic amino-acid interactions and a proline kink involving the highly conserved NPXXY motif. In phospholipid bicelles, TMH7 structure, orientation, and topology relative to H8 are dynamically modulated by the surrounding membrane phospholipid bilayer. These data provide a contextual basis for the present solid-state NMR study to investigate whether intermolecular interactions between hCB1(TMH7/H8) and its phospholipid environment may affect membrane-bilayer structure. For this purpose, we measured 1H–13C heteronuclear dipolar couplings for the choline, glycerol, and acyl-chain regions of dimyristoylphosphocholine in a magnetically aligned hCB1(TMH7/H8) bicelle sample. The results identify discrete regional interactions between hCB1(TMH7/H8) and membrane lipid molecules that increase phospholipid motion and decrease phospholipid order, indicating that the peptide’s partial traversal of the bilayer alters membrane structure. These data offer new insight into hCB1(TMH7/H8) properties and support the concept that the membrane bilayer itself may serve as a mechanochemical mediator of hCB1/GPCR signal transduction. Since interaction with its membrane environment has been implicated in hCB1 function and its modulation by small-molecule therapeutics, our work should help inform hCB1 pharmacology and the design of hCB1-targeted drugs