Gunnar Kleinau

Over the past decade, numerous studies have shown for several adhesion GPCRs (aGPCRs) that signaling activity is regulated by an intramolecular agonist (termed Stachel ) with a conserved amino acid core located proximal to the N terminus of transmembrane helix 1. Here we present cryo-EM structures of the human ADGRG6 (or GPR126) C-terminal fragment bound to heterotrimeric Gs protein in two distinct conformational states — an “ECL2-in” state that sterically and allosterically limits Stachel engagement and a “ Stachel -bound” active state with a core orientation unknown so far — determined to global resolutions of 3.4 Å and 3.8 Å, respectively. In the “ECL2-in” state the central segment of this loop makes a network of hydrogen-bonding and van-der-Waals contacts with the transmembrane domain. Of note, the conserved TM3–ECL2 cysteines do not form the canonical disulfide bridge in the “ECL2-in” conformation. Structure-guided mutagenesis, peptide stimulation and cellular signaling assays map inactivating and constitutively activating variants to spatially distinct ECL2 regions. Together, these data establish ECL2 as an intramolecular, tunable gate that controls internal-agonist engagement and G-protein activation in GPR126, revealing a structural mechanism that can be exploited to bias aGPCR signaling and might support new ideas for rational ligand design.

Human trichromatic color vision relies on three cone opsins [long-, middle-, and short-wavelength-sensitive opsins (LWS-, MWS-, and SWS-opsins, respectively)], whereas scotopic rod vision is mediated by rhodopsin. Although the structure of rhodopsin was solved more than 20 years ago, cone opsin structures have been lacking. Here, we present cryo–electron microscopy structures of the three human cone opsins, each bound to a G protein and all- trans retinal in the presumed active state. All three cone opsins differ markedly from rhodopsin. Within the retinal binding pocket, we identified a distinct counterion site (LWS- and MWS-opsins) and a ring of serines around the retinal (SWS-opsin). The active cone opsin structures explain how amino acid substitutions fine-tune spectral sensitivity and help clarify the molecular basis of color vision deficiencies and key differences in rod versus cone activation.

The domestic chicken exhibits extraordinarily high plumage diversity in sharp contrast to the uniform plumage phenotype in its wild ancestor, the red junglefowl. The melanocortin-1 receptor ( MC1R ) locus is the most variable pigmentation locus in domestic chicken. Here we have analyzed whole-genome sequence data from 10,026 birds, including red junglefowls and 393 populations of domestic chicken. We document ultrarapid evolution of 18 MC1R alleles, due to the accumulation of 9 missense mutations combined with reshuffling of sequence variants within the single MC1R exon. The lack of linkage disequilibrium at the MC1R locus strongly suggests that this combinatorial allele diversity is generated by interallelic gene conversion, possibly promoted by its high guanine–cytosine content. Functional analyses of MC1R variants expressed in transfected cells, using second-messenger and bioluminescence resonance energy transfer assays together with homology modeling, demonstrate that most missense mutations have significant effects on MC1R signaling and include both activating and inhibitory changes. One common MC1R allele differs by as many as three functionally relevant missense mutations from the most closely related wild-type allele, and its phenotype reflects their combined effects. Genome-wide analyses further show that MC1R acts as a modifier of dominant white and recessive white phenotypes caused by mutations in premelanosome protein and tyrosinase , respectively. In both cases, MC1R alleles promote a pure white phenotype, either by enhancing eumelanin pigmentation (dominant white) or inhibiting eumelanin pigmentation (recessive white).

The domestication of plants and animals has resulted in one of the most significant cultural and socio‐economical transitions in human history. Domestication of animals, including human‐supervised reproduction, largely uncoupled particular animal species from their natural, evolutionary history driven by environmental and ecological factors. The primary motivations for domesticating animals were, and still are, producing food and materials (e.g. meat, eggs, honey or milk products, wool, leather products, jewelry and medication products) to support plowing in agriculture or in transportation (e.g. horse, cattle, camel and llama) and to facilitate human activities (for hunting, rescuing, therapeutic aid, guarding behavior and protecting or just as a companion). In recent years, decoded genetic information from more than 40 domesticated animal species have become available; these studies have identified genes and mutations associated with specific physiological and behavioral traits contributing to the complex genetic background of animal domestication. These breeding‐altered genomes provide insights into the regulation of different physiological areas, including information on links between e.g. endocrinology and behavior, with important pathophysiological implications (e.g. for obesity and cancer), extending the interest in domestication well beyond the field. Several genes that have undergone selection during domestication and breeding encode specific G protein‐coupled receptors, a class of membrane‐spanning receptors involved in the regulation of a number of overarching functions such as reproduction, development, body homeostasis, metabolism, stress responses, cognition, learning and memory. Here we summarize the available literature on variations in G protein‐coupled receptors and their ligands and how these have contributed to animal domestication.

Functional melanocortin receptor (MCR) genes have been identified in the genomes of early chordates, e.g., the cyclostomata. Whether they appear in the most ancient chordates such as cephalochordate and urochordata, however, remains unclear due to missing genetic data. Herein, we studied five putative (from NCBI database), sequence-based predicted MCR-like receptors from urochordata and cephalochordate, including Styela clava, Ciona intestinalis, Branchiostoma floridae, and Branchiostoma belcheri. The BLAST and phylogenetic analyses suggested a relationship between these specific receptors and vertebrate MCRs. However, several essential residues for MCR functions in vertebrates were missing in these putative chordata MCRs. To test receptor functionality, several experimental studies were conducted. Binding assays and functional analyses showed no specific binding and no ligand-induced cAMP or ERK1/2 signaling (with either endogenous α-MSH or synthetic ligands for MC4R), despite successfully expressing four receptors in HEK 293T cells. These four receptors showed high basal cAMP signaling, likely mediated by ligand-independent Gs coupling. In summary, our results suggest that the five predicted MCR-like receptors are, indeed, class A G protein-coupled receptors (GPCRs), which in four cases show high constitutive activity in the Gs-cAMP signaling pathway but are not MCR-like receptors in terms of ligand recognition of known MCR ligands. These receptors might be ancient G protein-coupled receptors with so far unidentified ligands.

The melanocortin-4 receptor (MC4R) is a key player in the hypothalamic leptin–melanocortin pathway that regulates satiety and hunger. MC4R belongs to the G protein-coupled receptors (GPCRs), which are known to form heterodimers with other membrane proteins, potentially modulating receptor function or characteristics. Like MC4R, thyroid hormones (TH) are also essential for energy homeostasis control. TH transport across membranes is facilitated by the monocarboxylate transporter 8 (MCT8), which is also known to form heterodimers with GPCRs. Based on the finding in single-cell RNA-sequencing data that both proteins are simultaneously expressed in hypothalamic neurons, we investigated a putative interplay between MC4R and MCT8. We developed a novel staining protocol utilizing a fluorophore-labeled MC4R ligand and demonstrated a co-localization of MC4R and MCT8 in human brain tissue. Using in vitro assays such as BRET, IP1, and cAMP determination, we found that MCT8 modulates MC4R-mediated phospholipase C activation but not cAMP formation via a direct interaction, an effect that does not require a functional MCT8 as it was not altered by a specific MCT8 inhibitor. This suggests an extended functional spectrum of MCT8 as a GPCR signaling modulator and argues for the investigation of further GPCR-protein interactions with hitherto underrepresented physiological functions.

The clinical spectrum of thyrotropin receptor–mediated (TSHR-mediated) diseases varies from loss-of-function mutations causing congenital hypothyroidism to constitutively active mutations (CAMs) leading to nonautoimmune hyperthyroidism (NAH). Variation at the TSHR locus has also been associated with altered lipid and bone metabolism and autoimmune thyroid diseases. However, the extrathyroidal roles of TSHR and the mechanisms underlying phenotypic variability among TSHR-mediated diseases remain unclear. Here we identified and characterized TSHR variants and factors involved in phenotypic variability in different patient cohorts, the FinnGen database, and a mouse model. TSHR CAMs were found in all 16 patients with NAH, with 1 CAM in an unexpected location in the extracellular leucine-rich repeat domain (p.S237N) and another in the transmembrane domain (p.I640V) in 2 families with distinct hyperthyroid phenotypes. In addition, screening of the FinnGen database revealed rare functional variants as well as distinct common noncoding TSHR SNPs significantly associated with thyroid phenotypes, but there was no other significant association between TSHR variants and more than 2,000 nonthyroid disease endpoints. Finally, our TSHR M453T–knockin model revealed that the phenotype was dependent on the mutation’s signaling properties and was ameliorated by increased iodine intake. In summary, our data show that TSHR-mediated disease risk can be modified by variants at the TSHR locus both inside and outside the coding region as well as by altered TSHR-signaling and dietary iodine, supporting the need for personalized treatment strategies.

G protein-coupled receptor 83 (GPR83) is a class A G protein-coupled receptor with predominant expression in the cerebellum and proposed function in the regulation of food intake and in anxiety-like behavior. The neuropeptide PEN has been suggested as a specific GPR83 ligand. However, conflicting reports exist about whether PEN is indeed able to bind and activate GPR83. This study was initiated to evaluate PEN as a potential ligand of GPR83. Employing several second messenger and other GPCR activation assays as well as a radioligand binding assay, and using multiple GPR83 plasmids and PEN peptides from different sources, no experimental evidence was found to support a role of PEN as a GPR83 ligand.

G protein-coupled receptor 83 (GPR83) is a class A G protein-coupled receptor with predominant expression in the cerebellum and proposed function in the regulation of food intake and in anxiety-like behavior. The neuropeptide PEN has been suggested as a specific GPR83 ligand. However, conflicting reports exist about whether PEN is indeed able to bind and activate GPR83. This study was initiated to evaluate PEN as a potential ligand of GPR83. Employing several second-messenger and other GPCR activation assays as well as a radioligand binding assay, and using multiple GPR83 plasmids and PEN peptides from different sources, no experimental evidence was found to support a role of PEN as a GPR83 ligand.