Deciphering the role of glycosaminoglycans in GPCR signaling.

G protein-coupled receptors (GPCR) and glycosaminoglycans (GAGs) are two essential components of the cell surface that regulate physiological processes in the body. GPCRs are the most extensive family of transmembrane receptors that control cellular responses to extracellular stimuli, while GAGs are polysaccharides that contribute to the function of the extracellular matrix (ECM). Due to their proximity to the plasma membrane, GAGs participate in signal transduction by interacting with various extracellular molecules and cell surface receptors.

Altered Signaling and Desensitization Responses in PTH1R Mutants Associated with Eiken Syndrome.

The parathyroid hormone receptor type 1 (PTH1R) is a G protein-coupled receptor that plays key roles in regulating calcium homeostasis and skeletal development via binding the ligands, PTH and PTH-related protein (PTHrP), respectively. Eiken syndrome is a rare disease of delayed bone mineralization caused by homozygous PTH1R mutations. Of the three mutations identified so far, R485X, truncates the PTH1R C-terminal tail, while E35K and Y134S alter residues in the receptor's amino-terminal extracellular domain.

Molecular Mechanisms of PTH/PTHrP Class B GPCR Signaling and Pharmacological Implications.

The classical paradigm of G protein-coupled receptor (GPCR) signaling via G proteins is grounded in a view that downstream responses are relatively transient and confined to the cell surface, but this notion has been revised in recent years following the identification of several receptors that engage in sustained signaling responses from subcellular compartments following internalization of the ligand-receptor complex. This phenomenon was initially discovered for the parathyroid hormone (PTH) type 1 receptor (PTH1R), a vital GPCR for maintaining normal calcium and phosphate levels in the body with the paradoxical ability to build or break down bone in response to PTH binding. The diverse biological processes regulated by this receptor are thought to depend on its capacity to mediate diverse modes of cyclic adenosine monophosphate (cAMP) signaling.

Biased GPCR signaling by the native parathyroid hormone-related protein 1 to 141 relative to its N-terminal fragment 1 to 36.

The parathyroid hormone (PTH)-related protein (PTHrP) is indispensable for the development of mammary glands, placental calcium ion transport, tooth eruption, bone formation and bone remodeling, and causes hypercalcemia in patients with malignancy. Although mature forms of PTHrP in the body consist of splice variants of 139, 141, and 173 amino acids, our current understanding on how endogenous PTHrP transduces signals through its cognate G-protein coupled receptor (GPCR), the PTH type 1 receptor (PTHR), is largely derived from studies done with its N-terminal fragment, PTHrP. Here, we demonstrate using various fluorescence imaging approaches at the single cell level to measure kinetics of (i) receptor activation, (ii) receptor signaling via Gs and Gq, and (iii) receptor internalization and recycling that the native PTHrP displays biased agonist signaling properties that are not mimicked by PTHrP.

Precise druggability of the PTH type 1 receptor.

Class B G protein-coupled receptors (GPCRs) are notoriously difficult to target by small molecules because their large orthosteric peptide-binding pocket embedded deep within the transmembrane domain limits the identification and development of nonpeptide small molecule ligands. Using the parathyroid hormone type 1 receptor (PTHR) as a prototypic class B GPCR target, and a combination of molecular dynamics simulations and elastic network model-based methods, we demonstrate that PTHR druggability can be effectively addressed. Here we found a key mechanical site that modulates the collective dynamics of the receptor and used this ensemble of PTHR conformers to identify selective small molecules with strong negative allosteric and biased properties for PTHR signaling in cell and PTH actions in vivo.

Spatial bias in cAMP generation determines biological responses to PTH type 1 receptor activation.

The parathyroid hormone (PTH) type 1 receptor (PTHR) is a class B G protein–coupled receptor (GPCR) that regulates mineral ion, vitamin D, and bone homeostasis. Activation of the PTHR by PTH induces both transient cell surface and sustained endosomal cAMP production. To address whether the spatial (location) or temporal (duration) dimension of PTHR-induced cAMP encodes distinct biological outcomes, we engineered a biased PTHR ligand (PTH) that elicits cAMP production at the plasma membrane but not at endosomes.

Shining light on location-biased cAMP signaling.

cAMP is the indispensable second messenger regulating cell metabolism and function in response to extracellular hormones and neurotransmitters. cAMP is produced via the activation of G protein-coupled receptors located at both the cell surface and inside the cell. Recently, Tsvetanova et al.

Ligand-Dependent Effects of Methionine-8 Oxidation in Parathyroid Hormone Peptide Analogues.

LA-PTH is a long-acting parathyroid hormone (PTH) peptide analogue in preclinical development for hypoparathyroidism (HP). Like native PTH, LA-PTH contains a methionine at position 8 (Met8) that is predicted to be critical for function. We assessed the impact of Met oxidation on the functional properties of LA-PTH and control PTH ligands.

PTH hypersecretion triggered by a GABA and Ca-sensing receptor heterocomplex in hyperparathyroidism.

Molecular mechanisms mediating tonic secretion of parathyroid hormone (PTH) in response to hypocalcaemia and hyperparathyroidism (HPT) are unclear. Here we demonstrate increased heterocomplex formation between the calcium-sensing receptor (CaSR) and metabotropic γ-aminobutyric acid (GABA) B receptor (GABAR) in hyperplastic parathyroid glands (PTGs) of patients with primary and secondary HPT. Targeted ablation of GABAR or glutamic acid decarboxylase 1 and 2 in PTGs produces hypocalcaemia and hypoparathyroidism, and prevents PTH hypersecretion in PTGs cultured from mouse models of hereditary HPT and dietary calcium-deficiency.

Allosteric interactions in the parathyroid hormone GPCR-arrestin complex formation.

Peptide ligands of class B G-protein-coupled receptors act via a two-step binding process, but the essential mechanisms that link their extracellular binding to intracellular receptor-arrestin interactions are not fully understood. Using NMR, crosslinking coupled to mass spectrometry, signaling experiments and computational approaches on the parathyroid hormone (PTH) type 1 receptor (PTHR), we show that initial binding of the PTH C-terminal part constrains the conformation of the flexible PTH N-terminal signaling epitope before a second binding event occurs. A 'hot-spot' PTH residue, His9, that inserts into the PTHR transmembrane domain at this second step allosterically engages receptor-arrestin coupling.

Structural insights into emergent signaling modes of G protein-coupled receptors.

G protein-coupled receptors (GPCRs) represent the largest family of cell membrane proteins, with >800 GPCRs in humans alone, and recognize highly diverse ligands, ranging from photons to large protein molecules. Very important to human medicine, GPCRs are targeted by about 35% of prescription drugs. GPCRs are characterized by a seven-transmembrane α-helical structure, transmitting extracellular signals into cells to regulate major physiological processes via heterotrimeric G proteins and β-arrestins.

G-dependent regulation of endosomal cAMP generation by parathyroid hormone class B GPCR.

cAMP production upon activation of G by G protein-coupled receptors has classically been considered to be plasma membrane-delimited, but a shift in this paradigm has occurred in recent years with the identification of several receptors that continue to signal from early endosomes after internalization. The molecular mechanisms regulating this aspect of signaling remain incompletely understood. Here, we investigated the role of G activation by the parathyroid hormone (PTH) type 1 receptor (PTHR) in mediating endosomal cAMP responses.

PTH/PTHrP Receptor Signaling, Allostery, and Structures.

The parathyroid hormone (PTH) type 1 receptor (PTHR) is the canonical G protein-coupled receptor (GPCR) for PTH and PTH-related protein (PTHrP) and the key regulator of calcium homeostasis and bone turnover. PTHR function is critical for human health to maintain homeostatic control of ionized serum Ca levels and has several unusual signaling features, such as endosomal cAMP signaling, that are well-studied but not structurally understood. In this review, we discuss how recently solved high resolution near-atomic structures of hormone-bound PTHR in its inactive and active signaling states and discovery of extracellular Ca allosterism shed light on the structural basis for PTHR signaling and function.

Use of Backbone Modification To Enlarge the Spatiotemporal Diversity of Parathyroid Hormone Receptor-1 Signaling via Biased Agonism.

The type-1 parathyroid hormone receptor (PTHR1), which regulates calcium homeostasis and tissue development, has two native agonists, parathyroid hormone (PTH) and PTH-related protein (PTHrP). PTH forms a complex with the PTHR1 that is rapidly internalized and induces prolonged cAMP production from endosomes. In contrast, PTHrP induces only transient cAMP production, which primarily arises from receptors on the cell surface.

Structure and dynamics of the active human parathyroid hormone receptor-1.

The parathyroid hormone receptor-1 (PTH1R) is a class B G protein-coupled receptor central to calcium homeostasis and a therapeutic target for osteoporosis and hypoparathyroidism. Here we report the cryo-electron microscopy structure of human PTH1R bound to a long-acting PTH analog and the stimulatory G protein. The bound peptide adopts an extended helix with its amino terminus inserted deeply into the receptor transmembrane domain (TMD), which leads to partial unwinding of the carboxyl terminus of transmembrane helix 6 and induces a sharp kink at the middle of this helix to allow the receptor to couple with G protein.

Ca allostery in PTH-receptor signaling.

The parathyroid hormone (PTH) and its related peptide (PTHrP) activate PTH receptor (PTHR) signaling, but only the PTH sustains G-mediated adenosine 3',5'-cyclic monophosphate (cAMP) production after PTHR internalization into early endosomes. The mechanism of this unexpected behavior for a G-protein-coupled receptor is not fully understood. Here, we show that extracellular Ca acts as a positive allosteric modulator of PTHR signaling that regulates sustained cAMP production.

Luminescence-activated nucleotide cyclase regulates spatial and temporal cAMP synthesis.

cAMP is a ubiquitous second messenger that regulates cellular proliferation, differentiation, attachment, migration, and several other processes. It has become increasingly evident that tight regulation of cAMP accumulation and localization confers divergent yet specific signaling to downstream pathways. Currently, few tools are available that have sufficient spatial and temporal resolution to study location-biased cAMP signaling.

Cdc42 activation couples fluid shear stress to apical endocytosis in proximal tubule cells.

Cells lining the kidney proximal tubule (PT) respond to acute changes in glomerular filtration rate and the accompanying fluid shear stress (FSS) to regulate reabsorption of ions, glucose, and other filtered molecules and maintain glomerulotubular balance. Recently, we discovered that exposure of PT cells to FSS also stimulates an increase in apical endocytic capacity (Raghavan et al. PNAS, 111:8506-8511, 2014).

Dual role of mitochondria in producing melatonin and driving GPCR signaling to block cytochrome c release.

G protein-coupled receptors (GPCRs) are classically characterized as cell-surface receptors transmitting extracellular signals into cells. Here we show that central components of a GPCR signaling system comprised of the melatonin type 1 receptor (MT), its associated G protein, and β-arrestins are on and within neuronal mitochondria. We discovered that the ligand melatonin is exclusively synthesized in the mitochondrial matrix and released by the organelle activating the mitochondrial MT signal-transduction pathway inhibiting stress-mediated cytochrome release and caspase activation.

Design and validation of the first cell-impermeant melatonin receptor agonist.

Background And Purpose: The paradigm that GPCRs are able to prolong or initiate cellular signalling through intracellular receptors recently emerged. Melatonin binds to G protein-coupled MT and MT receptors. In contrast to most other hormones targeting GPCRs, melatonin and its synthetic analogues are amphiphilic molecules easily penetrating into cells, but the existence of intracellular receptors is still unclear mainly due to a lack of appropriate tools.

β-adrenergic receptor control of endosomal PTH receptor signaling via Gβγ.

Cells express several G-protein-coupled receptors (GPCRs) at their surfaces, transmitting simultaneous extracellular hormonal and chemical signals into cells. A comprehensive understanding of mechanisms underlying the integrated signaling response induced by distinct GPCRs is thus required. Here we found that the β-adrenergic receptor, which induces a short cAMP response, prolongs nuclear cAMP and protein kinase A (PKA) activation by promoting endosomal cAMP production in parathyroid hormone (PTH) receptor signaling through the stimulatory action of G protein Gβγ subunits on adenylate cyclase type 2.