Ligand recognition and G-protein coupling selectivity of cholecystokinin A receptor

Cholecystokinin A receptor (CCKAR) belongs to family A G-protein-coupled receptors and regulates nutrient homeostasis upon stimulation by cholecystokinin (CCK). It is an attractive drug target for gastrointestinal and metabolic diseases. One distinguishing feature of CCKAR is its ability to interact with a sulfated ligand and to couple with divergent G-protein subtypes, including Gs, Gi and Gq. However, the basis for G-protein coupling promiscuity and ligand recognition by CCKAR remains unknown. Here, we present three cryo-electron microscopy structures of sulfated CCK-8-activated CCKAR in complex with Gs, Gi and Gq heterotrimers, respectively. CCKAR presents a similar conformation in the three structures, whereas conformational differences in the ‘wavy hook’ of the Gα subunits and ICL3 of the receptor serve as determinants in G-protein coupling selectivity. Our findings provide a framework for understanding G-protein coupling promiscuity by CCKAR and uncover the mechanism of receptor recognition by sulfated CCK-8.

Receptor-wide Determinants of G Protein Coupling Selectivity in Aminergic GPCRs

G protein-coupled receptors (GPCRs) induce signal transduction pathways through coupling to four main subtypes of G proteins (Gs, Gi, Gq, G12/13), selectively. However, G protein selective activation mechanisms and residual determinants in GPCRs have remained obscure. Here, we identified conserved G protein selective activation mechanisms determining receptors’ ability to couple to a type of G protein. Herein, we performed an extensive phylogenetic analysis and identified specifically conserved residues for the receptors having similar coupling profiles in each aminergic receptor. By integrating our methodology of differential evolutionary conservation of G protein-specific amino acids with structural analyses, we identified selective activation networks for Gs, Gi1, Go, and Gq. We found that G protein selectivity is determined by not only the G protein interaction site but also other parts of the receptor including the ligand binding pocket. To validate our findings, we further studied an amino acid residue that we revealed as a selectivity-determining in Gs coupling and performed molecular dynamics (MD) simulations. We showed that previously uncharacterized Glycine at position 7×41 plays an important role in both receptor activation and Gs coupling. Finally, we gathered our results into a comprehensive model of G protein selectivity called “sequential switches of activation” describing three main molecular switches controlling GPCR activation: ligand binding, G protein selective activation mechanisms and G protein contact. We believe that our work provides a broader view on receptor-level determinants of G protein coupling selectivity.

Structural basis for ligand recognition and G protein-coupling promiscuity of the cholecystokinin A receptor

Cholecystokinin A receptor (CCKAR) belongs to family A G protein-coupled receptors (GPCRs) and regulates nutrient homeostasis upon stimulation by cholecystokinin (CCK). It is an attractive drug target for gastrointestinal and metabolic diseases. One distinguishing feature of CCKAR is its ability to interact with sulfated ligand and to couple with divergent G protein subtypes, including Gs, Gi, and Gq. However, the basis for G protein coupling promiscuity and ligand recognition by CCKAR remain unknown. Here we present three cryo-electron microscopy (cryo-EM) structures of sulfated CCK-8 activated CCKAR in complex with Gs, Gi, and Gq heterotrimers, respectively. In these three structures, CCKAR presents a similar conformation, whereas conformational differences in ″wavy hook″ of Gα subunits and ICL3 of the receptor serve as determinants in G protein coupling selectivity. These structures together with mutagenesis data provide the framework for understanding the G protein coupling promiscuity by CCKAR and uncover the mechanism of receptor recognition by sulfated CCK-8.

Constitutive G protein coupling profiles of understudied orphan GPCRs

A large number of GPCRs are potentially valuable drug targets but remain understudied. Many of these lack well-validated activating ligands and are considered “orphan” receptors, and G protein coupling profiles have not been defined for many orphan GPCRs. Here we asked if constitutive receptor activity can be used to determine G protein coupling profiles of orphan GPCRs. We monitored nucleotide-sensitive interactions between 48 understudied orphan GPCRs and five G proteins (240 combinations) using bioluminescence resonance energy transfer (BRET). No receptor ligands were used, but GDP was used as a common G protein ligand to disrupt receptor-G protein complexes. Constitutive BRET between the same receptors and β-arrestins was also measured. We found sufficient GDP-sensitive BRET to generate G protein coupling profiles for 22 of the 48 receptors we studied. Altogether we identified 48 coupled receptor-G protein pairs, many of which have not been described previously. We conclude that receptor-G protein complexes that form spontaneously in the absence of guanine nucleotides can be used to profile G protein coupling of constitutively-active GPCRs. This approach may prove useful for studying G protein coupling of other GPCRs for which activating ligands are not available.