The Orphan G Protein-Coupled Receptor GPR52 is a Novel Regulator of Breast Cancer Multicellular Organization.

Statement Of Significance: We showed that loss of the orphan G protein-coupled receptor GPR52 in human breast cell lines leads to increased cell clustering, hybrid/partial EMT, and increased tumor burden in zebrafish.

Background: G protein-coupled receptors (GPCRs) are the largest class of membrane-bound receptors that transmit critical signals from extracellular to intracellular spaces. Transcriptomic data of resected breast tumors show that low mRNA expression of orphan GPCR GPR52 correlates with reduced overall survival in patients with breast cancer, leading to the hypothesis that loss of GPR52 supports breast cancer progression.

KCTD Proteins Have Redundant Functions in Controlling Cellular Growth.

We explored the functional redundancy of three structurally related KCTD (Potassium Channel Tetramerization Domain) proteins, KCTD2, KCTD5, and KCTD17, by progressively knocking them out in HEK 293 cells using CRISPR/Cas9 genome editing. After validating the knockout, we assessed the effects of progressive knockout on cell growth and gene expression. We noted that the progressive effects of knockout of KCTD isoforms on cell growth were most pervasive when all three isoforms were deleted, suggesting some functions were conserved between them.

Structure and dynamics of a pentameric KCTD5/CUL3/Gβγ E3 ubiquitin ligase complex.

Heterotrimeric G proteins can be regulated by posttranslational modifications, including ubiquitylation. KCTD5, a pentameric substrate receptor protein consisting of an N-terminal BTB domain and a C-terminal domain, engages CUL3 to form the central scaffold of a cullin-RING E3 ligase complex (CRL3) that ubiquitylates Gβγ and reduces Gβγ protein levels in cells. The cryo-EM structure of a 5:5:5 KCTD5/CUL3/Gβγ assembly reveals a highly dynamic complex with rotations of over 60° between the KCTD5/CUL3 and KCTD5/Gβγ moieties of the structure.

Evidence for heterodimerization and functional interaction of the urotensin II and the angiotensin II type 1 receptors.

Despite the observation of synergistic interactions between the urotensinergic and angiotensinergic systems, the interplay between the urotensin II receptor (hUT) and the angiotensin II type 1 receptor (hAT1R) in regulating cellular signaling remains incompletely understood. Notably, the putative interaction between hUT and hAT1R could engender reciprocal allosteric modulation of their signaling signatures, defining a unique role for these complexes in cardiovascular physiology and pathophysiology. Using a combination of co-immunoprecipitation, bioluminescence resonance energy transfer (BRET) and FlAsH BRET-based conformational biosensors, we first demonstrated the physical interaction between hUT and hAT1R.

Allosteric modulation of GPCR-induced β-arrestin trafficking and signaling by a synthetic intrabody.

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a primary determinant of β-arrestin (βarr) recruitment and trafficking. For several GPCRs such as the vasopressin receptor subtype 2 (VR), agonist-stimulation first drives the translocation of βarrs to the plasma membrane, followed by endosomal trafficking, which is generally considered to be orchestrated by multiple phosphorylation sites. We have previously shown that mutation of a single phosphorylation site in the VR (i.

IBD-associated G protein-coupled receptor 65 variant compromises signalling and impairs key functions involved in inflammation.

Background And Aims: Inflammatory bowel diseases (IBD) result in chronic inflammation of the gastrointestinal tract. Genetic studies have shown that the GPR65 gene, as well as its missense coding variant, GPR65*Ile231Leu, is associated with IBD. We aimed to define the signalling and biological pathways downstream of GPR65 activation and evaluate the impact of GPR65*231Leu on these.

Addition of a carboxy-terminal tail to the normally tailless gonadotropin-releasing hormone receptor impairs fertility in female mice.

Gonadotropin-releasing hormone (GnRH) is the primary neuropeptide controlling reproduction in vertebrates. GnRH stimulates follicle-stimulating hormone (FSH) and luteinizing hormone (LH) synthesis via a G-protein-coupled receptor, GnRHR, in the pituitary gland. In mammals, GnRHR lacks a C-terminal cytosolic tail (Ctail) and does not exhibit homologous desensitization.

The MAP kinase ERK5/MAPK7 is a downstream effector of oxytocin signaling in myometrial cells.

The hormone oxytocin (OT) has pleiotropic activities both in the central nervous system as well as in peripheral tissues, including uterotonic effects on the myometrium during parturition. OT effects are mediated by a single transmembrane receptor, belonging to the GPCR (G protein-coupled receptor) superfamily and coupled primarily to Gq- and Gi-containing heterotrimeric G proteins. Upon receptor stimulation, one well-studied downstream effect is activation of the ERK1/2 MAP (mitogen-activated protein) kinase, and studies have shown that induction of COX-2 by OT in the myometrium required ERK1/2 activity.

Signal profiling of the βAR reveals coupling to novel signalling pathways and distinct phenotypic responses mediated by βAR and βAR.

A comprehensive understanding of signalling downstream of GPCRs requires a broad approach to capture novel signalling modalities in addition to established pathways. Here, using an array of sixteen validated BRET-based biosensors, we analyzed the ability of seven different β-adrenergic ligands to engage five distinct signalling pathways downstream of the β-adrenergic receptor (βAR). In addition to generating signalling signatures and capturing functional selectivity for the different ligands toward these pathways, we also revealed coupling to signalling pathways that have not previously been ascribed to the βAR.

Design and biological assessment of membrane-tethering neuroprotective peptides derived from the pituitary adenylate cyclase-activating polypeptide type 1 receptor.

Background: The pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1), a class B G protein-coupled receptor (GPCR), has emerged as a promising target for treating neurodegenerative conditions. Unfortunately, despite years of research, no PAC1-specific agonist has been discovered, as activity on two other GPCRs, VPAC1 and VPAC2, is retained with current analogs. Cell signaling is related to structural modifications in the intracellular loops (ICLs) of GPCRs.

Combining Conformational Profiling of GPCRs with CRISPR/Cas9 Gene Editing Approaches.

Ligand-biased signaling could have a significant impact on drug discovery programs. As such, many approaches to screening now target a larger section of the signaling responses downstream of an individual G protein-coupled receptor (GPCR). Biosensor-based platforms have been developed to capture signaling signatures.

Asymmetric Recruitment of β-Arrestin1/2 by the Angiotensin II Type I and Prostaglandin F2α Receptor Dimer.

Initially identified as monomers, G protein-coupled receptors (GPCRs) can also form functional homo- and heterodimers that act as distinct signaling hubs for cellular signal integration. We previously found that the angiotensin II (Ang II) type 1 receptor (AT1R) and the prostaglandin F2α (PGF2α) receptor (FP), both important in the control of smooth muscle contractility, form such a functional heterodimeric complex in HEK 293 and vascular smooth muscle cells. Here, we hypothesize that both Ang II- and PGF2α-induced activation of the AT1R/FP dimer, or the parent receptors alone, differentially regulate signaling by distinct patterns of β-arrestin recruitment.

Measuring Recruitment of β-Arrestin to G Protein-Coupled Heterodimers Using Bioluminescence Resonance Energy Transfer.

Initially identified as monomers, G protein-coupled receptors (GPCRs) can also form functional dimers that act as distinct signalling hubs for the integration of cellular signalling. We previously found that the angiotensin II (Ang II) type 1 receptor (AT1R) and the prostaglandin F2α (PGF2α) receptor (FP), both important in the control of smooth muscle contractility, form such a functional heterodimeric complex in HEK 293 and vascular smooth muscle cells (Goupil et al., J Biol Chem 290:3137-3148, 2015; Sleno et al.

Functional selectivity profiling of the angiotensin II type 1 receptor using pathway-wide BRET signaling sensors.

G protein-coupled receptors (GPCRs) are important therapeutic targets that exhibit functional selectivity (biased signaling), in which different ligands or receptor variants elicit distinct downstream signaling. Understanding all the signaling events and biases that contribute to both the beneficial and adverse effects of GPCR stimulation by given ligands is important for drug discovery. Here, we report the design, validation, and use of pathway-selective bioluminescence resonance energy transfer (BRET) biosensors that monitor the engagement and activation of signaling effectors downstream of G proteins, including protein kinase C (PKC), phospholipase C (PLC), p63RhoGEF, and Rho.

BRET-based assay to monitor EGFR transactivation by the ATR reveals G protein-independent activation and ATR-EGFR complexes.

The type 1 angiotensin II (AngII) receptor (ATR) transactivates the epidermal growth factor receptor (EGFR), which leads to pathological remodeling of heart, blood vessels and kidney. End-point assays are used as surrogates of EGFR activation, however these downstream readouts are not applicable to live cells, in real-time. Herein, we report the use of a bioluminescence resonance energy transfer (BRET)-based assay to assess recruitment of the EGFR adaptor protein, growth factor receptor-bound protein 2 (Grb2), to the EGFR.

New insights about the peculiar role of the 28-38 C-terminal segment and some selected residues in PACAP for signaling and neuroprotection.

The pituitary adenylate cyclase-activating polypeptide (PACAP), which exists in two isoforms of 27 and 38 amino acids, can induce neuronal protection in vitro and in vivo following the activation of PAC1, a class B G protein-coupled receptor (GPCR). With its potent neuroprotective and anti-inflammatory effects, this peptide represents a promising avenue for the development of therapeutic strategies to potentially cure or at least slow the progression of neurodegenerative disorders. Beyond the canonical G protein signal effectors, GPCRs are also coupled to a multitude of intracellular signaling pathways that can be independently activated by biased ligands, thereby expanding vastly the potential for discovering new drugs.

Conformational biosensors reveal allosteric interactions between heterodimeric AT1 angiotensin and prostaglandin F2α receptors.

G protein-coupled receptors (GPCRs) are conformationally dynamic proteins transmitting ligand-encoded signals in multiple ways. This transmission is highly complex and achieved through induction of distinct GPCR conformations, which preferentially drive specific receptor-mediated signaling events. This conformational capacity can be further enlarged via allosteric effects between dimers, warranting further study of these effects.

Distinct Conformational Dynamics of Three G Protein-Coupled Receptors Measured Using FlAsH-BRET Biosensors.

A number of studies have profiled G protein-coupled receptor (GPCR) conformation using fluorescent biaresenical hairpin binders (FlAsH) as acceptors for BRET or FRET. These conformation-sensitive biosensors allow reporting of movements occurring on the intracellular surface of a receptor to investigate mechanisms of receptor activation and function. Here, we generated eight FlAsH-BRET-based biosensors within the sequence of the β-adrenergic receptor (βAR) and compared agonist-induced responses to the angiotensin II receptor type I (AT1R) and the prostaglandin F2α receptor (FP).

Conformational Profiling of the AT1 Angiotensin II Receptor Reflects Biased Agonism, G Protein Coupling, and Cellular Context.

Here, we report the design and use of G protein-coupled receptor-based biosensors to monitor ligand-mediated conformational changes in receptors in intact cells. These biosensors use bioluminescence resonance energy transfer with luciferase (RlucII) as an energy donor, placed at the distal end of the receptor C-tail, and the small fluorescent molecule FlAsH as an energy acceptor, its binding site inserted at different positions throughout the intracellular loops and C-terminal tail of the angiotensin II type I receptor. We verified that the modifications did not compromise receptor localization or function before proceeding further.

Cellular and subcellular context determine outputs from signaling biosensors.

The use of biosensors either individually or as part of panels has now become a common technique to capturing signaling events in living cells. Such biosensors have become particularly important for probing biased signaling and allostery in G protein-coupled receptor drug screening efforts. However, assumptions about the portability of such biosensors between cell types may lead to misinterpretation of drug effects on specific signaling pathways in a given cellular context.

Tracking GPCR biosynthesis and degradation using a nonradioactive pulse chase methodology.

The β2-adrenergic receptor (β2AR) is a prototypical member of the G protein-coupled receptor (GPCR) superfamily of proteins and is one of the best-characterized GPCRs due to its role in several important physiological systems. Because of limited availability of high quality antibodies against GPCRs, much of the work done on β2AR took advantage of heterologous expression systems. Overexpressed proteins may overwhelm the cellular regulatory machinery leading potentially to responses distinct from the native protein.

Dual allosteric modulation of opioid antinociceptive potency by α2A-adrenoceptors.

Opioid and α2-adrenoceptor (AR) agonists are analgesic when administered in the spinal cord and show a clinically beneficial synergistic interaction when co-administered. However, α2-AR antagonists can also inhibit opioid antinociception, suggesting a complex interaction between the two systems. The α2A-AR subtype is necessary for spinal adrenergic analgesia and synergy with opioids for most agonist combinations.

Designing BRET-based conformational biosensors for G protein-coupled receptors.

Ligand-biased signaling is starting to have significant impact on drug discovery programs in the pharmaceutical industry and has reinvigorated our understanding of pharmacological efficacy. As such, many investigators and screening campaigns are now being directed at a larger section of the signaling responses downstream of an individual G protein-coupled receptor. Many biosensor-based platforms have been developed to capture signaling signatures.