Cryo-EM Unlocks GPCR Secrets: Advances in Endocrinology, Metabolism & Drug Discovery

Abstract

G protein-coupled receptors represent the largest class of cell surface receptors and are of crucial importance in signal transduction, endocrinology, and metabolism. In the last ten years, cryogenic electron microscopy has brought a truly revolutionary change to the study of GPCR structures, thus revolutionizing unprecedented insights into their functioning and therapeutic opportunities. Cryo-EM resolution of the first GPCR structures in 2017 has rapidly gained over X-ray crystallography. Today, more than 650 GPCR structures have been visualized with cryo-EM. This article will discuss important structural features of GPCRs revealed through cryo-EM regarding ligand recognition, receptor activation, G protein coupling, and arrestin recruitment. In this regard, we also highlight allosteric binding sites as having a potentiating role in biased signaling, which, in turn, might determine selective activation of certain pathways. We mention examples involving glycoprotein hormone receptors and the glucagon-like peptide 1 receptor to demonstrate how cryo-EM has advanced GPCR biology in endocrinology and metabolism. This review captures the transformative impact of cryo-EM for GPCR research and has wide implications in drug discovery and endocrine disease treatment.

Introduction

The largest family of cell surface receptors, G protein-coupled receptors have the most diverse members and participate in many physiological processes: signal transduction, endocrine functions, and metabolic regulation. Encoding within the human genome more than 800 GPCRs, is pivotal in the modulation of hormonal responses, neurotransmission, immune reactions, and sensory perception. G Protein-Coupled Receptors have long been under biomedical research due to their critical roles in human health. About 30-40% of all approved drugs target GPCRs, thus their drug discovery relevance is exceptional.

Unlocking the therapeutic potential of GPCRs requires an understanding of their molecular architecture. Until recently, in the early 2000s, X-ray crystallography was the only real methodology to progress GPCR structures; however, cryogenic electron microscopy, or cryo-EM has dramatically changed the landscape of structural biology. As cryo-EM realized its first GPCR structures in 2017, this technology has now overtaken X-ray crystallography in terms of both quality and number of GPCR structures. Since 2023, over 650 GPCR structures have been disclosed using cryo-EM with the count growing exponentially; we will review structural characteristics of GPCRs revealed by cryo-EM, briefly discuss the impact of cryo-EM on our ligand recognition, receptor activation, and biased signaling, and highlight its contribution to advancing the research in endocrinology and metabolism.

The Structural Revolution of GPCRs

The Cryo-EM Breakthrough

Cryo-EM has revolutionized the field of structural biology. It now offers near-atomic resolution of proteins without the requirement of crystallization. This technique involves extremely rapid freezing of biological samples in a thin layer of vitreous ice that keeps the native structure of the sample. The 2D projection images of the sample are captured with electron beams and then computationally synthesized into 3D models.

The very first pair of GPCR structures resolved by cryo-EM in 2017 broke that landmark over the limitations of X-ray crystallography, which was unable to handle the dynamic and flexible nature of GPCRs. Such early successes paved the way for a huge explosion of GPCR structural data figures for doubling cryo-EM-resolved GPCRs every 0.63 years. Today, cryo-EM is the standard tool in GPCR research, which provides unexpected insights into their complex structures and functions.

Advantages of Cryo-EM Over X-ray Crystallography

Cryo-EM offers several key advantages over X-ray crystallography

No Need for Crystallization: X-ray crystallography requires protein crystals, which are difficult to obtain for GPCRs due to their flexibility and membrane-bound nature. Cryo-EM circumvents this challenge by imaging GPCRs in their native environment.

Capture of Dynamic Conformations: GPCRs are highly dynamic, existing in multiple conformational states. Cryo-EM allows researchers to capture these different states, providing a more comprehensive view of GPCR activation and signaling.

Complexes with Large Proteins: Cryo-EM is particularly well-suited for studying large GPCR complexes, including those with G proteins, arrestins, or other signaling partners.

GPCR Structure and Function

GPCRs are seven transmembrane (7TM) helical structures that it used as a scaffold for ligand binding, receptor activation, and intracellular signaling. Altogether, these receptors account for interaction with virtually the full range of ligands including hormones, neurotransmitters, and sensory molecules, to mention a few that trigger cellular responses.

Ligand Recognition

The most important function of GPCRs is the recognition of extracellular ligands that alter receptor conformation. Thus, the structural determinants of ligand binding have been understood by Cryo-EM to reveal heterogeneity in the GPCR binding pocket. Some key findings include:

Orthosteric and Allosteric Sites: GPCRs have both orthosteric (primary) and allosteric (secondary) binding sites. Cryo-EM has helped identify how ligands bind to these distinct regions, offering potential targets for drug development.

Biased Ligand Binding: Biased ligands preferentially activate specific signaling pathways (e.g., G protein vs. arrestin pathways), which could lead to more selective and effective therapies. Cryo-EM has revealed how subtle changes in ligand structure can dictate biased signaling, providing a roadmap for designing drugs with fewer side effects.

Receptor Activation and G Protein Coupling

GPCRs undergo conformational changes upon ligand binding, which allow them to couple with intracellular G proteins. Cryo-EM has been instrumental in capturing these activated states, providing a detailed view of how GPCRs transmit signals across the cell membrane.

Conformational Changes: Cryo-EM has shown that ligand binding induces structural rearrangements in the 7TM helices, which in turn create a binding site for the G protein. These conformational changes are critical for initiating intracellular signaling.

G Protein Coupling: Cryo-EM studies have resolved the structures of GPCR-G protein complexes, shedding light on how different GPCRs preferentially couple with specific G proteins (e.g., Gs, Gi, Gq). Understanding these interactions is crucial for developing drugs that target specific signaling pathways.

Arrestin Recruitment and Regulation by GPCR Kinases

In addition to G protein coupling, interaction between arrestins and GPCRs also leads to receptor desensitization and internalization. Cryo-EM structures of GPCR-arrestin complexes have given the most detailed views into how these interactions modulate receptor activity and signal transduction.

GPCR Kinases (GRKs): GRKs phosphorylate GPCRs, which increases the affinity for arrestin binding. Cryo-EM revealed how GRKs recognize and phosphorylate GPCRs and shed light on receptor signaling regulation.

Allosteric Modulation and Biased Signaling

Allosteric modulation is defined as the regulation of GPCR activity with the help of ligands that bind to other sites rather than the primary orthosteric binding site. Therefore, these allosteric ligands may provide either activation or inhibition of the activity of the receptors, thus offering a more selective approach to drug design.

Diversity of Allosteric Sites

Cryo-EM has revealed a variety of allosteric binding sites in GPCRs that opens the door to designing allosteric modulators. Compared to orthosteric sites, they are most often less conserved, making drugs that would be much more selective for certain receptors easier to create.

Biased Signaling

Biased signaling occurs when a ligand preferentially activates one signaling pathway over another. For example, a biased ligand might selectively activate G protein signaling while avoiding arrestin-mediated desensitization. Cryo-EM has revealed the structural basis of biased signaling, showing how different ligands stabilize specific GPCR conformations.

Impact of Cryo-EM on Endocrinology and Metabolism

GPCRs play crucial roles in endocrinology and metabolism, regulating hormone responses, glucose homeostasis, and lipid metabolism. Cryo-EM has provided new insights into the structural biology of endocrine-related GPCRs, with implications for understanding diseases like diabetes, obesity, and thyroid disorders.

Glycoprotein Hormone Receptors

Cryo-EM studies of glycoprotein hormone receptors, such as the thyroid-stimulating hormone (TSH) receptor and follicle-stimulating hormone (FSH) receptor, have revealed the mechanisms of hormone recognition and receptor activation. These findings have important implications for treating endocrine disorders such as hyperthyroidism and infertility.

Glucagon-like peptide 1 (GLP-1) Receptor

The GLP-1 receptor is a critical regulator of glucose metabolism and a key target for diabetes treatment. Cryo-EM has provided detailed structures of the GLP-1 receptor in complex with GLP-1 analogs, offering new insights into the development of antidiabetic drugs.

Cryo-EM and Drug Discovery

The explosion of GPCR structural data generated by cryo-EM has transformed drug discovery, enabling the design of more selective and potent therapeutics. By providing high-resolution structures of GPCRs in complex ligands, cryo-EM has facilitated the rational design of drugs that target specific receptor subtypes or signaling pathways.

Structure-Based Drug Design: Cryo-EM allows for the visualization of drug-receptor interactions at atomic resolution, enabling the design of molecules that fit precisely into GPCR binding pockets.

Allosteric Modulators: The discovery of allosteric sites on GPCRs has opened up new avenues for drug development. Allosteric modulators offer the potential for greater specificity and fewer side effects compared to traditional orthosteric drugs.

Conclusion

Cryo-EM has revolutionized our perspective on GPCR structure and function by unlocking hitherto unprecedented insight into ligand recognition, receptor activation, and biased signaling. Its influence on endocrinology and metabolism, through the power of drug discovery, has completely changed the game and has unleashed new targeted therapies for the treatment of vast ranges of diseases. Further advancements with cryo-EM technology will unlock the full therapeutic potential of GPCRs and revolutionize the next-generation treatment approach for endocrine and metabolic disorders.

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