Regulation of G Protein Signaling by GoLoco Motif Containing Proteins

Signal transduction via heterotrimeric G proteins in response to transmembrane G protein-coupled receptors plays a central aspect in how cells integrate extracellular stimuli and produce biological responses. In addition to receptor-mediated activation of heterotrimeric G proteins, during the last few decades, accessory proteins have been found to regulate G protein activity via different mechanisms. Several proteins have been identified that contain multiple G protein regulatory domains. Using various molecular and biochemical approaches, we have characterized the effects of two such proteins, G18 and RGS14, on G protein activity. Both proteins contain a second G protein binding domain in addition to the GoLoco domain, which primarily acts as a guanine nucleotide dissociation inhibitor (GDI) on Gi/o proteins. Our results identified that the N-terminal region of G18 is a novel G protein-interacting domain, which may have distinct regulatory effects within the Gi/o subfamily and could potentially play a role in differentiating signals between these related G proteins. In addition, we characterized the tissue and cellular distribution of G18. We found that G18 is expressed in primary isolated rat aortic smooth muscle cells and endothelial cells. A protein-protein interaction assay indicated that G18 is able to directly interact with RGS5, an RGS protein that is also highly expressed in vascular tissue. This interaction results in an increase in RGS5 GTPase accelerating protein (GAP) activity with little or no effect on G18 activity. In Chapter 4 of the thesis, we identified a novel GAP enhancing domain located at the Ras-binding (RB) region of RGS14. This enhancement may be due to the intramolecular interaction between the RB domain and the RGS domain. Furthermore, this interaction may also result in an inhibitory effect on the GDI activity of the RGS14 GoLoco motif. Overall, my work suggests that GoLoco motif containing proteins G18 and RGS14 are organizers of G protein signaling that also modulate RGS protein function

Structural and functional diversity among agonist-bound states of the GLP-1 receptor

Recent advances in G-protein-coupled receptor (GPCR) structural elucidation have strengthened previous hypotheses that multidimensional signal propagation mediated by these receptors depends, in part, on their conformational mobility; however, the relationship between receptor function and static structures is inherently uncertain. Here, we examine the contribution of peptide agonist conformational plasticity to activation of the glucagon-like peptide 1 receptor (GLP-1R), an important clinical target. We use variants of the peptides GLP-1 and exendin-4 (Ex4) to explore the interplay between helical propensity near the agonist N terminus and the ability to bind to and activate the receptor. Cryo-EM analysis of a complex involving an Ex4 analog, the GLP-1R and Gs heterotrimer revealed two receptor conformers with distinct modes of peptide-receptor engagement. Our functional and structural data, along with molecular dynamics (MD) simulations, suggest that receptor conformational dynamics associated with flexibility of the peptide N-terminal activation domain may be a key determinant of agonist efficacy.</p

Understanding VPAC receptor family peptide binding and selectivity

The vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) receptors are key regulators of neurological processes. Despite recent structural data, a comprehensive understanding of peptide binding and selectivity among different subfamily receptors is lacking. Here, we determine structures of active, Gs-coupled, VIP-VPAC1R, PACAP27-VPAC1R, and PACAP27-PAC1R complexes. Cryo-EM structural analyses and molecular dynamics simulations (MDSs) reveal fewer stable interactions between VPAC1R and VIP than for PACAP27, more extensive dynamics of VIP interaction with extracellular loop 3, and receptor-dependent differences in interactions of conserved N-terminal peptide residues with the receptor core. MD of VIP modelled into PAC1R predicts more transient VIP-PAC1R interactions in the receptor core, compared to VIP-VPAC1R, which may underlie the selectivity of VIP for VPAC1R over PAC1R. Collectively, our work improves molecular understanding of peptide engagement with the PAC1R and VPAC1R that may benefit the development of novel selective agonists

Dynamics of GLP-1R peptide agonist engagement are correlated with kinetics of G protein activation

The glucagon-like peptide-1 receptor (GLP-1R) has broad physiological roles and is a validated target for treatment of metabolic disorders. Despite recent advances in GLP-1R structure elucidation, detailed mechanistic understanding of how different peptides generate profound differences in G protein-mediated signalling is still lacking. Here we combine cryo-electron microscopy, molecular dynamics simulations, receptor mutagenesis and pharmacological assays, to interrogate the mechanism and consequences of GLP-1R binding to four peptide agonists; glucagon-like peptide-1, oxyntomodulin, exendin-4 and exendin-P5. These data reveal that distinctions in peptide N-terminal interactions and dynamics with the GLP-1R transmembrane domain are reciprocally associated with differences in the allosteric coupling to G proteins. In particular, transient interactions with residues at the base of the binding cavity correlate with enhanced kinetics for G protein activation, providing a rationale for differences in G protein-mediated signalling efficacy from distinct agonists

Activation of the GLP-1 receptor by a non-peptidic agonist

Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, including diabetes and obesity1. Structures of active receptors reveal peptide agonists engage deep within the receptor core, leading to an outward movement of extracellular loop 3 and the tops of transmembrane helices 6 and 7, an inward movement of transmembrane helix 1, reorganization of extracellular loop 2 and outward movement of the intracellular side of transmembrane helix 6, resulting in G-protein interaction and activation2,3,4,5,6. Here we solved the structure of a non-peptide agonist, TT-OAD2, bound to the glucagon-like peptide-1 (GLP-1) receptor. Our structure identified an unpredicted non-peptide agonist-binding pocket in which reorganization of extracellular loop 3 and transmembrane helices 6 and 7 manifests independently of direct ligand interaction within the deep transmembrane domain pocket. TT-OAD2 exhibits biased agonism, and kinetics of G-protein activation and signalling that are distinct from peptide agonists. Within the structure, TT-OAD2 protrudes beyond the receptor core to interact with the lipid or detergent, providing an explanation for the distinct activation kinetics that may contribute to the clinical efficacy of this compound series. This work alters our understanding of the events that drive the activation of class B receptors