Structure, function and pharmacology of human itch GPCRs.

The MRGPRX family of receptors (MRGPRX1-4) is a family of mas-related G-protein-coupled receptors that have evolved relatively recently1. Of these, MRGPRX2 and MRGPRX4 are key physiological and pathological mediators of itch and related mast cell-mediated hypersensitivity reactions2-5. MRGPRX2 couples to both Gi and Gq in mast cells6. Here we describe agonist-stabilized structures of MRGPRX2 coupled to Gi1 and Gq in ternary complexes with the endogenous peptide cortistatin-14 and with a synthetic agonist probe, respectively, and the development of potent antagonist probes for MRGPRX2. We also describe a specific MRGPRX4 agonist and the structure of this agonist in a complex with MRGPRX4 and Gq. Together, these findings should accelerate the structure-guided discovery of therapeutic agents for pain, itch and mast cell-mediated hypersensitivity.

Structures of the human cholecystokinin 1 (CCK1) receptor bound to Gs and Gq mimetic proteins provide insight into mechanisms of G protein selectivity.

G protein-coupled receptors (GPCRs) are critical regulators of cellular function acting via heterotrimeric G proteins as their primary transducers with individual GPCRs capable of pleiotropic coupling to multiple G proteins. Structural features governing G protein selectivity and promiscuity are currently unclear. Here, we used cryo-electron microscopy (cryo-EM) to determine structures of the cholecystokinin (CCK) type 1 receptor (CCK1R) bound to the CCK peptide agonist, CCK-8 and 2 distinct transducer proteins, its primary transducer Gq, and the more weakly coupled Gs. As seen with other Gq/11-GPCR complexes, the Gq-α5 helix (αH5) bound to a relatively narrow pocket in the CCK1R core. Surprisingly, the backbone of the CCK1R and volume of the G protein binding pocket were essentially equivalent when Gs was bound, with the Gs αH5 displaying a conformation that arises from "unwinding" of the far carboxyl-terminal residues, compared to canonically Gs coupled receptors. Thus, integrated changes in the conformations of both the receptor and G protein are likely to play critical roles in the promiscuous coupling of individual GPCRs.

Structures in G proteins important for subtype selective receptor binding and subsequent activation.

G protein-coupled receptors (GPCRs) selectively couple to specific heterotrimeric G proteins comprised of four subfamilies in order to induce appropriate physiological responses. However, structural determinants in Gα subunits responsible for selective recognition by approximately 800 human GPCRs have remained elusive. Here, we directly compare the influence of subtype-specific Gα structures on the stability of GPCR-G protein complexes and the activation by two Gq-coupled receptors. We used FRET-assays designed to distinguish multiple Go and Gq-based Gα chimeras in their ability to be selectively bound and activated by muscarinic M3 and histaminic H1 receptors. We identify the N-terminus including the αN/ß1-hinge, the ß2/ß3-loop and the α5 helix of Gα to be key selectivity determinants which differ in their impact on selective binding to GPCRs and subsequent activation depending on the specific receptor. Altogether, these findings provide new insights into the molecular basis of G protein-coupling selectivity even beyond the Gα C-terminus.

Ultrastructure and localization of a visual Gq protein in hypertrophied epitoke ocelli of Perinereis brevicirris (Polychaeta, Annelida).

Functional ultrastructural changes in the rhabdomeric photoreceptors of the cerebral ocelli are described for normal and sexually mature (epitoke) Perinereis brevicirris (Polychaeta, Annelida). With sexual maturation, the cerebral ocelli hypertrophied, increasing in volume to 5.5 times that of ocelli in the normal state, and the thickness of the retinal layer increased up to 10 times. Perinereis ocelli have a pigmented retinal layer consisting of at least two cell types: photoreceptor cell (PR) and pigmented supporting cells (PS). In epitoke ocelli, PR bear well-developed rhabdomeric microvilli, multilamellar bodies, and numerous cytoplasmic membranous structures, including vesicles, smooth endoplasmic reticulum, and secondary lysosomes. Localization of a visual Gq protein in the ocelli was studied with anti-GqC antibody. The antibody strongly labeled not only microvilli and multilamellar bodies throughout the retinal layer, but also secondary lysosomes and vesicles in the cytoplasm of the PR in the epitoke ocelli, although labeling was observed only in the microvilli and multilamellar bodies in normal ocelli. Reverse transcription/polymerase chain reaction analysis revealed that the amount of G protein alpha subunit mRNA in the epitoke head increased by roughly twice that of the normal head. Since Gq protein is essential for phototransduction in Perinereis ocelli, these results suggest that the sites are involved in photoreceptive membrane turnover, which occurs much more extensively in epitoke ocelli. Thus, epitoke ocelli may represent a model system for studying rhabdomeric photoreceptive membrane turnover.