RESUMEN
Like other secreted peptides, nascent parathyroid hormone (PTH) is synthesized with a pre- and a pro-sequence (25 and 6 amino acids, respectively). These precursor segments are sequentially removed in parathyroid cells before packaging into secretory granules. Three patients from two unrelated families who presented during infancy with symptomatic hypocalcemia were found to have a homozygous serine (S) to proline (P) change affecting the first amino acid of the mature PTH. Unexpectedly, biological activity of synthetic [P1]PTH(1-34) was indistinguishable from that of unmodified [S1]PTH(1-34). However, in contrast to conditioned medium from COS-7 cells expressing prepro[S1]PTH(1-84), medium from cells expressing prepro[P1]PTH(1-84) failed to stimulate cAMP production despite similar PTH levels when measured by an intact assay that detects PTH(1-84) and large amino-terminally truncated fragments thereof. Analysis of the secreted, but inactive PTH variant led to the identification of pro[P1]PTH(-6 to +84). Synthetic pro[P1]PTH(-6 to +34) and pro[S1]PTH(-6 to +34) had much less bioactivity than the corresponding PTH(1-34) analogs. Unlike pro[S1]PTH(-6 to +34), pro[P1]PTH(-6 to +34) was resistant to cleavage by furin suggesting that the amino acid variant impairs preproPTH processing. Consistent with this conclusion, plasma of patients with the homozygous P1 mutation had elevated proPTH levels, as determined with an in-house assay specific for pro[P1]PTH(-6 to +84). In fact, a large fraction of PTH detected by the commercial intact assay represented the secreted pro[P1]PTH. In contrast, two commercial biointact assays that use antibodies directed against the first few amino acid residues of PTH(1-84) for capture or detection failed to detect pro[P1]PTH.
Asunto(s)
Hipocalcemia , Humanos , Hipocalcemia/genética , Hormona Paratiroidea/genética , Hormona Paratiroidea/metabolismo , Mutación , Prolina/genética , Aminoácidos/genéticaRESUMEN
The parathyroid hormone type 1 receptor (PTHR1), a Class B GPCR, is activated by long polypeptides, including drugs for osteoporosis and hypoparathyroidism. The PTHR1 engages peptide agonists via a two-step mechanism. Initial contact involves the extracellular domain (ECD), which has been thought to contribute primarily to receptor-peptide binding, and then the N terminus of the peptide engages the receptor transmembrane domain (TMD), which is thought to control the message conveyed to intracellular partners. This mechanism has been suggested to apply to other Class B GPCRs as well. Here, we show that modification of a PTHR1 agonist at ECD-contact sites can alter the signaling profile, an outcome that is not accommodated by the current two-step binding model. Our data support a modified two-step binding model in which agonist orientation on the ECD surface can influence the geometry of agonist-TMD engagement. This expanded binding model offers a mechanism by which altering ECD-contact residues can affect signaling profile. Our discoveries provide a rationale for exploring agonist modifications distal from the TMD-contact region in future efforts to optimize therapeutic performance of peptide hormone analogs.
Asunto(s)
Receptor de Hormona Paratiroídea Tipo 1 , Transducción de Señal , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , Unión Proteica , Dominios Proteicos , Péptidos/metabolismoRESUMEN
Parathyroid hormone 1 receptor (PTH1R) plays a key role in mediating calcium homeostasis and bone development, and aberrant PTH1R activity underlies several human diseases. Peptidic PTH1R antagonists and inverse agonists have therapeutic potential in treating these diseases, but their poor pharmacokinetics and pharmacodynamics undermine their in vivo efficacy. Herein, we report the use of a backbone-modification strategy to design a peptidic PTH1R inhibitor that displays prolonged activity as an antagonist of wild-type PTH1R and an inverse agonist of the constitutively active PTH1R-H223R mutant both in vitro and in vivo. This peptide may be of interest for the future development of therapeutic agents that ameliorate PTH1R malfunction.
Asunto(s)
Agonismo Inverso de Drogas , Receptor de Hormona Paratiroídea Tipo 1 , Humanos , Péptidos , Hormona Paratiroidea/farmacologíaRESUMEN
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. This study provides a computational pipeline to detect precise druggable sites and identify allosteric modulators of PTHR signaling that could be extended to GPCRs to expedite discoveries of small molecules as novel therapeutic candidates.
Asunto(s)
Receptor de Hormona Paratiroídea Tipo 1 , Receptores Acoplados a Proteínas G , Ligandos , Simulación de Dinámica Molecular , Transducción de SeñalRESUMEN
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, PTHrP1-36. 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 PTHrP1-141 displays biased agonist signaling properties that are not mimicked by PTHrP1-36. Although PTHrP1-36 induces transient cAMP production, acute intracellular Ca2+ (iCa2+) release and ß-arrestin recruitment mediated by ligand-PTHR interactions at the plasma membrane, PTHrP1-141 triggers sustained cAMP signaling from the plasma membrane and fails to stimulate iCa2+ release and recruit ß-arrestin. Furthermore, we show that the molecular basis for biased signaling differences between PTHrP1-36 and properties of native PTHrP1-141 are caused by the stabilization of a singular PTHR conformation and PTHrP1-141 sensitivity to heparin, a sulfated glycosaminoglycan. Taken together, our results contribute to a better understanding of the biased signaling process of a native protein hormone acting in conjunction with a GPCR.
Asunto(s)
Receptor de Hormona Paratiroídea Tipo 1 , AMP Cíclico/metabolismo , Heparina/metabolismo , Humanos , Ligandos , Conformación Proteica , Receptor de Hormona Paratiroídea Tipo 1/química , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , Transducción de Señal , beta-Arrestinas/metabolismoRESUMEN
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. A conformational change in PTHR intracellular loop 3 permits favorable interactions with ß-arrestin's finger loop. These results unveil structural determinants for PTHR-arrestin complex formation and reveal that the two-step binding mechanism proceeds via cooperative fluctuations between ligand and receptor, which extend to other class B G-protein-coupled receptors.
Asunto(s)
Arrestina/metabolismo , Hormona Paratiroidea/metabolismo , Arrestina/química , Fosfatos de Calcio , Microscopía por Crioelectrón , AMP Cíclico , Escherichia coli , Células HEK293 , Humanos , Simulación de Dinámica Molecular , Hormona Paratiroidea/química , Receptores Acoplados a Proteínas GRESUMEN
Human parathyroid hormone (PTH) and N-terminal fragments thereof activate two receptors, hPTHR1 and hPTHR2, which share â¼51% sequence similarity. A peptide comprising the first 34 residues of PTH is fully active at both receptors and is used to treat osteoporosis. We have used this system to explore the hypothesis that backbone modification of a promiscuous peptidic agonist can provide novel receptor-selective agonists. We tested this hypothesis by preparing a set of variants of PTH(1-34)-NH2 that contained a single ß-amino-acid residue replacement at each of the first eight positions. These homologs, each containing one additional backbone methylene unit relative to PTH(1-34)-NH2 itself, displayed a wide range of potencies in cell-based assays for PTHR1 or PTHR2 activation. The ß-scan series allowed us to identify two homologs, each containing two αâß replacements, that were highly selective, one for PTHR1 and the other for PTHR2. These findings suggest that backbone modification of peptides may provide a general strategy for achieving activation selectivity among polypeptide-modulated receptors, and that success requires consideration of both ß2- and ß3-residues, which differ in terms of side-chain location.
Asunto(s)
Hormona Paratiroidea/química , Péptidos/síntesis química , Péptidos/farmacología , Receptor de Hormona Paratiroídea Tipo 1/agonistas , Receptor de Hormona Paratiroídea Tipo 2/agonistas , Secuencia de Aminoácidos , Células HEK293 , Humanos , Unión Proteica , Conformación ProteicaRESUMEN
Osteoporosis can result from the loss of sex hormones and/or aging. Abaloparatide (ABL), an analog of parathyroid hormone-related protein (PTHrP(1-36)), is the second osteoanabolic therapy approved by the United States Food and Drug Administration after teriparatide (PTH(1-34)). All three peptides bind PTH/PTHrP receptor type 1 (PTHR1), but the effects of PTHrP(1-36) or ABL in the osteoblast remain unclear. We show that, in primary calvarial osteoblasts, PTH(1-34) promotes a more robust cAMP response than PTHrP(1-36) and ABL and causes a greater activation of protein kinase A (PKA) and cAMP response element-binding protein (CREB). All three peptides similarly inhibited sclerostin (Sost). Interestingly, the three peptides differentially modulated two other PKA target genes, c-Fos and receptor activator of NF-κB ligand (Rankl), and the latter both in vitro and in vivo Knockdown of salt-inducible kinases (SIKs) 2 and 3 and CREB-regulated transcription coactivator 3 (CRTC3), indicated that all three are part of the pathway that regulates osteoblastic Rankl expression. We also show that the peptides differentially regulate the nuclear localization of CRTC2 and CRTC3, and that this correlates with PKA activation. Moreover, inhibition of protein phosphatases 1 and 2A (PP1/PP2A) activity revealed that they play a major role in both PTH-induced Rankl expression and the effects of PTH(1-34) on CRTC3 localization. In summary, in the osteoblast, the effects of PTH(1-34), PTHrP(1-36), and ABL on Rankl are mediated by differential stimulation of cAMP/PKA signaling and by their downstream effects on SIK2 and -3, PP1/PP2A, and CRTC3.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Osteoblastos/metabolismo , Hormona Paratiroidea , Proteína Fosfatasa 1/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ligando RANK/biosíntesis , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismo , Animales , Línea Celular Tumoral , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Ratones , Osteoblastos/citología , Hormona Paratiroidea/análogos & derivados , Hormona Paratiroidea/farmacología , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 2/genética , Proteínas Serina-Treonina Quinasas/genética , Ligando RANK/genética , Ratas , Transducción de Señal/genética , Factores de Transcripción/genéticaRESUMEN
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. We show that backbone modification of PTH(1-34)-NH2 and abaloparatide (a PTHrP derivative) with a single homologous ß-amino acid residue can generate biased agonists that induce prolonged cAMP production from receptors at the cell surface. This unique spatiotemporal profile could be useful for distinguishing effects associated with the duration of cAMP production from effects associated with the site of cAMP production.
Asunto(s)
Receptor de Hormona Paratiroídea Tipo 1/química , Secuencia de Aminoácidos , AMP Cíclico/biosíntesis , Células HEK293 , Humanos , Proteína Relacionada con la Hormona Paratiroidea/farmacología , Receptor de Hormona Paratiroídea Tipo 1/agonistas , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
Peptide agonists of GPCRs and other receptors are powerful signaling molecules with high potential as biological tools and therapeutics, but they are typically plagued by instability and short half-lives in vivo. Nature uses protein glycosylation to increase the serum stability of secreted proteins. However, these extracellular modifications are complex and heterogeneous in structure, making them an impractical solution. In contrast, intracellular proteins are subjected to a simple version of glycosylation termed O-GlcNAc modification. In our studies of this modification, we found that O-GlcNAcylation inhibits proteolysis, and strikingly, this stabilization occurs despite large distances in primary sequence (10-15 amino acids) between the O-GlcNAc and the site of cleavage. We therefore hypothesized that this "remote stabilization" concept could be useful to engineer the stability and potentially additional properties of peptide or protein therapeutics. Here, we describe the application of O-GlcNAcylation to two clinically important peptides: glucagon-like peptide-1 (GLP-1) and the parathyroid hormone (PTH), which respectively help control glucose and calcium levels in the blood. For both peptides, we found O-GlcNAcylated analogs that are equipotent to unmodified peptide in cell-based activation assays, while several GLP-1 analogs were biased agonists relative to GLP-1. As we predicted, O-GlcNAcylation can improve the stability of both GLP-1 and PTH in serum despite the fact that the O-GlcNAc can be quite remote from characterized sites of peptide cleavage. The O-GlcNAcylated GLP-1 and PTH also displayed significantly improved in vivo activity. Finally, we employed structure-based molecular modeling and receptor mutagenesis to predict how O-GlcNAcylation can be accommodated by the receptors and the potential interactions that contribute to peptide activity. This approach demonstrates the potential of O-GlcNAcylation for generating analogs of therapeutic peptides with enhanced proteolytic stability.
Asunto(s)
Péptido 1 Similar al Glucagón/farmacología , Hormona Paratiroidea/farmacología , Ingeniería de Proteínas , Receptores Acoplados a Proteínas G/agonistas , Péptido 1 Similar al Glucagón/sangre , Péptido 1 Similar al Glucagón/química , Glicosilación , Humanos , Hormona Paratiroidea/sangre , Hormona Paratiroidea/química , Conformación Proteica , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
The type-1 parathyroid hormone receptor (PTHR1) is a family B G protein-coupled receptor (GPCR) that mediates the actions of two polypeptide ligands; parathyroid hormone (PTH), an endocrine hormone that regulates the levels of calcium and inorganic phosphate in the blood by acting on bone and kidney, and PTH-related protein (PTHrP), a paracrine-factor that regulates cell differentiation and proliferation programs in developing bone and other tissues. The type-2 parathyroid hormone receptor (PTHR2) binds a peptide ligand, called tuberoinfundibular peptide-39 (TIP39), and while the biologic role of the PTHR2/TIP39 system is not as defined as that of the PTHR1, it likely plays a role in the central nervous system as well as in spermatogenesis. Mechanisms of action at these receptors have been explored through a variety of pharmacological and biochemical approaches, and the data obtained support a basic "two-site" mode of ligand binding now thought to be used by each of the family B peptide hormone GPCRs. Recent crystallographic studies on the family B GPCRs are providing new insights that help to further refine the specifics of the overall receptor architecture and modes of ligand docking. One intriguing pharmacological finding for the PTHR1 is that it can form surprisingly stable complexes with certain PTH/PTHrP ligand analogs and thereby mediate markedly prolonged cell signaling responses that persist even when the bulk of the complexes are found in internalized vesicles. The PTHR1 thus appears to be able to activate the Gα(s)/cAMP pathway not only from the plasma membrane but also from the endosomal domain. The cumulative findings could have an impact on efforts to develop new drug therapies for the PTH receptors.
Asunto(s)
AMP Cíclico/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Modelos Moleculares , Receptores de Hormona Paratiroidea/metabolismo , Sistemas de Mensajero Secundario , Animales , Membrana Celular/enzimología , Membrana Celular/metabolismo , Endosomas/enzimología , Endosomas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Humanos , Agencias Internacionales , Ligandos , Farmacología/tendencias , Farmacología Clínica/tendencias , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/química , Isoformas de Proteínas/clasificación , Isoformas de Proteínas/metabolismo , Receptores de Hormona Paratiroidea/agonistas , Receptores de Hormona Paratiroidea/química , Receptores de Hormona Paratiroidea/clasificación , Sociedades Científicas , Terminología como AsuntoRESUMEN
It has been widely assumed that the production of the ubiquitous second messenger cyclic AMP, which is mediated by cell surface G proteincoupled receptors (GPCRs), and its termination take place exclusively at the plasma membrane. Recent studies reveal that diverse GPCRs do not always follow this conventional paradigm. In the new model, GPCRs mediate G-protein signaling not only from the plasma membrane but also from endosomal membranes. This model proposes that following ligand binding and activation, cell surface GPCRs internalize and redistribute into early endosomes, where trimeric G protein signaling can be maintained for an extended period of time. This Perspective discusses the molecular and cellular mechanistic subtleties as well as the physiological consequences of this unexpected process, which is considerably changing how we think about GPCR signaling and regulation and how we study drugs that target this receptor family.
Asunto(s)
AMP Cíclico/biosíntesis , Endosomas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Membrana Celular/metabolismo , Humanos , Receptores de Superficie Celular/metabolismo , Transducción de Señal/fisiologíaRESUMEN
The PTH receptor is to our knowledge one of the first G protein-coupled receptor (GPCR) found to sustain cAMP signaling after internalization of the ligand-receptor complex in endosomes. This unexpected model is adding a new dimension on how we think about GPCR signaling, but its mechanism is incompletely understood. We report here that endosomal acidification mediated by the PKA action on the v-ATPase provides a negative feedback mechanism by which endosomal receptor signaling is turned off.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Endosomas/metabolismo , Receptores Acoplados a Proteínas G/fisiología , Transducción de Señal/fisiología , ATPasas de Translocación de Protón Vacuolares/fisiología , Arrestinas/química , Arrestinas/metabolismo , Toxina del Cólera/farmacología , AMP Cíclico/fisiología , Retroalimentación Fisiológica , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Fosforilación , Unión Proteica , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , Receptor de Hormona Paratiroídea Tipo 1/fisiología , beta-ArrestinasRESUMEN
Agonist-induced phosphorylation of the parathyroid hormone (PTH) receptor 1 (PTHR1) regulates receptor signaling in vitro, but the role of this phosphorylation in vivo is uncertain. We investigated this role by injecting "knock-in" mice expressing a phosphorylation-deficient (PD) PTHR1 with PTH ligands and assessing acute biologic responses. Following injection with PTH (1-34), or with a unique, long-acting PTH analog, PD mice, compared with WT mice, exhibited enhanced increases in cAMP levels in the blood, as well as enhanced cAMP production and gene expression responses in bone and kidney tissue. Surprisingly, however, the hallmark hypercalcemic and hypophosphatemic responses were markedly absent in the PD mice, such that paradoxical hypocalcemic and hyperphosphatemic responses were observed, quite strikingly with the long-acting PTH analog. Spot urine analyses revealed a marked defect in the capacity of the PD mice to excrete phosphate, as well as cAMP, into the urine in response to PTH injection. This defect in renal excretion was associated with a severe, PTH-induced impairment in glomerular filtration, as assessed by the rate of FITC-inulin clearance from the blood, which, in turn, was explainable by an overly exuberant systemic hypotensive response. The overall findings demonstrate the importance in vivo of PTH-induced phosphorylation of the PTHR1 in regulating acute ligand responses, and they serve to focus attention on mechanisms that underlie the acute calcemic response to PTH and factors, such as blood phosphate levels, that influence it.
Asunto(s)
Huesos/metabolismo , Riñón/metabolismo , Hormona Paratiroidea/análogos & derivados , Receptor de Hormona Paratiroídea Tipo 1/fisiología , Animales , Calcio/sangre , Calcio/orina , AMP Cíclico/sangre , AMP Cíclico/orina , Relación Dosis-Respuesta a Droga , Perfilación de la Expresión Génica , Técnicas de Sustitución del Gen , Homeostasis , Humanos , Ligandos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfatos/sangre , Fosfatos/orina , Fosforilación , Ratas , Receptores Acoplados a Proteínas G/metabolismo , Factores de TiempoRESUMEN
Ligand-induced activation of G protein-coupled receptors (GPCRs) can initiate signaling through multiple distinct pathways with differing biological and physiological outcomes. There is intense interest in understanding how variation in GPCR ligand structure can be used to promote pathway selective signaling ("biased agonism") with the goal of promoting desirable responses and avoiding deleterious side effects. Here we present an approach in which a conventional peptide ligand for the type 1 parathyroid hormone receptor (PTHR1) is converted from an agonist which induces signaling through all relevant pathways to a compound that is highly selective for a single pathway. This is achieved not through variation in the core structure of the agonist, but rather by linking it to a nanobody tethering agent that binds with high affinity to a separate site on the receptor not involved in signal transduction. The resulting conjugate represents the most biased agonist of PTHR1 reported to date. This approach holds promise for facile generation of pathway selective ligands for other GPCRs.
Asunto(s)
Receptor de Hormona Paratiroídea Tipo 1 , Receptores Acoplados a Proteínas G , Transducción de Señal , Anticuerpos de Dominio Único , Ligandos , Humanos , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , Receptor de Hormona Paratiroídea Tipo 1/agonistas , Anticuerpos de Dominio Único/metabolismo , Anticuerpos de Dominio Único/farmacología , Células HEK293 , Transducción de Señal/efectos de los fármacos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Unión Proteica , Animales , Péptidos/química , Péptidos/farmacología , Péptidos/metabolismoRESUMEN
Osteocytes are the primary mechano-sensitive cell type in bone. Mechanical loading is sensed across the dendritic projections of osteocytes leading to transient reductions in focal adhesion kinase (FAK) activity. Knowledge regarding the signaling pathways downstream of FAK in osteocytes is incomplete. We performed tyrosine-focused phospho-proteomic profiling in osteocyte-like Ocy454 cells to identify FAK substrates. Gsα, parathyroid hormone receptor (PTH1R), and phosphodiesterase 8A (PDE8A), all proteins associated with cAMP signaling, were found as potential FAK targets based on their reduced tyrosine phosphorylation in both FAK- deficient or FAK inhibitor treated cells. Real time monitoring of intracellular cAMP levels revealed that FAK pharmacologic inhibition or gene deletion increased basal and GPCR ligand-stimulated cAMP levels and downstream phosphorylation of protein kinase A substrates. Mutating FAK phospho-acceptor sites in Gsα and PTH1R had no effect on PTH- or FAK inhibitor-stimulated cAMP levels. Since FAK inhibitor treatment augmented cAMP levels even in the presence of forskolin, we focused on potential FAK substrates downstream of cAMP generation. Indeed, PDE8A inhibition mimicked FAK inhibition at the level of increased cAMP, PKA activity, and expression of cAMP-regulated target genes. In vitro kinase assay showed that PDE8A is directly phosphorylated by FAK while immunoprecipitation assays revealed intracellular association between FAK and PDE8A. Thus, FAK inhibition in osteocytes acts synergistically with signals that activate adenylate cyclase to increase intracellular cAMP. Mechanically-regulated FAK can modulate intracellular cAMP levels via effects on PDE8A. These data suggest a novel signal transduction mechanism that mediates crosstalk between mechanical and cAMP-linked hormonal signaling in osteocytes.
RESUMEN
Vasopressin (VP) binds to the vasopressin type 2 receptor (V2R) to trigger physiological effects including body fluid homeostasis and blood pressure regulation. Signaling is terminated by receptor downregulation involving clathrin-mediated endocytosis and V2R degradation. We report here that both native and epitope-tagged V2R are internalized from the plasma membrane of LLC-PK1 kidney epithelial cells in the presence of another ligand, transferrin (Tf). The presence of iron-saturated Tf (holo-Tf; 4 h) reduced V2R binding sites at the cell surface by up to 33% while iron-free (apo-Tf) had no effect. However, no change in green fluorescent protein-tagged V2R distribution was observed in the presence of bovine serum albumin, atrial natriuretic peptide, or ANG II. Conversely, holo-Tf did not induce the internalization of another G protein-coupled receptor, the parathyroid hormone receptor. In contrast to the effect of VP, Tf did not increase intracellular cAMP or modify aquaporin-2 distribution in these cells, although addition of VP and Tf together augmented VP-induced V2R internalization. Tf receptor coimmunoprecipitated with V2R, suggesting that they interact closely, which may explain the additive effect of VP and Tf on V2R endocytosis. Furthermore, Tf-induced V2R internalization was abolished in cells expressing a dominant negative dynamin (K44A) mutant, indicating the involvement of clathrin-coated pits. We conclude that Tf can induce heterologous downregulation of the V2R and this might desensitize VP target cells without activating downstream V2R signaling events. It also provides new insights into urine-concentrating defects observed in rat models of hemochromatosis.
Asunto(s)
Regulación hacia Abajo/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Riñón/efectos de los fármacos , Receptores de Vasopresinas/metabolismo , Transferrina/farmacología , Vasopresinas/farmacología , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Endocitosis/efectos de los fármacos , Endocitosis/fisiología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Riñón/citología , Riñón/metabolismo , Receptores de Vasopresinas/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , PorcinosRESUMEN
The generation of cAMP by G protein-coupled receptors (GPCRs) and its termination are currently thought to occur exclusively at the plasma membrane of cells. Under existing models of receptor regulation, this signal is primarily restricted by desensitization of the receptors through their binding to ß-arrestins. However, this paradigm is not consistent with recent observations that the parathyroid hormone receptor type 1 (PTHR) continues to stimulate cAMP production even after receptor internalization, as ß-arrestins are known to rapidly bind and internalize activated PTHR. Here we show that binding to ß-arrestin1 prolongs rather than terminates the generation of cAMP by PTHR, and that cAMP generation correlates with the persistence of arrestin-receptor complexes on endosomes. PTHR signaling is instead turned off by the retromer complex, which regulates the movement of internalized receptor from endosomes to the Golgi apparatus. Thus, binding by the retromer complex regulates the sustained generation of cAMP triggered by an internalized GPCR.
Asunto(s)
AMP Cíclico/metabolismo , Endosomas/metabolismo , Aparato de Golgi/metabolismo , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , Arrestinas/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Modelos Biológicos , Receptor de Hormona Paratiroídea Tipo 1/genética , Transducción de Señal , Factores de Tiempo , beta-ArrestinasRESUMEN
Ligand-induced activation of G protein-coupled receptors (GPCRs) can initiate signaling through multiple distinct pathways with differing biological and physiological outcomes. There is intense interest in understanding how variation in GPCR ligand structure can be used to promote pathway selective signaling ("biased agonism") with the goal of promoting desirable responses and avoiding deleterious side effects. Here we present a new approach in which a conventional peptide ligand for the type 1 parathyroid hormone receptor (PTHR1) is converted from an agonist which induces signaling through all relevant pathways to a compound that is highly selective for a single pathway. This is achieved not through variation in the core structure of the agonist, but rather by linking it to a nanobody tethering agent that binds with high affinity to a separate site on the receptor not involved in signal transduction. The resulting conjugate represents the most biased agonist of PTHR1 reported to date. This approach holds promise for facile generation of pathway selective ligands for other GPCRs.