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1.
Mol Cell Endocrinol ; 589: 112235, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38621656

RESUMO

Luteinizing hormone (LH) is essential for reproduction, controlling ovulation and steroidogenesis. Its receptor (LHR) recruits various transducers leading to the activation of a complex signaling network. We recently identified iPRC1, the first variable fragment from heavy-chain-only antibody (VHH) interacting with intracellular loop 3 (ICL3) of the follicle-stimulating hormone receptor (FSHR). Because of the high sequence similarity of the human FSHR and LHR (LHCGR), here we examined the ability of the iPRC1 intra-VHH to modulate LHCGR activity. In this study, we demonstrated that iPRC1 binds LHCGR, to a greater extent when the receptor was stimulated by the hormone. In addition, it decreased LH-induced cAMP production, cAMP-responsive element-dependent transcription, progesterone and testosterone production. These impairments are not due to Gs nor ß-arrestin recruitment to the LHCGR. Consequently, iPRC1 is the first intra-VHH to bind and modulate LHCGR biological activity, including steroidogenesis. It should help further understand signaling mechanisms elicited at this receptor and their outcomes on reproduction.


Assuntos
Hormônio Luteinizante , Receptores do LH , Transdução de Sinais , Receptores do LH/metabolismo , Receptores do LH/genética , Humanos , Transdução de Sinais/efeitos dos fármacos , Hormônio Luteinizante/metabolismo , Animais , AMP Cíclico/metabolismo , Ligação Proteica , Progesterona/metabolismo , Receptores do FSH/metabolismo , Receptores do FSH/genética , Testosterona/metabolismo , Testosterona/biossíntese , Células HEK293 , Proteínas de Ligação ao GTP/metabolismo , Esteroides/biossíntese , Esteroides/metabolismo
3.
FEBS Lett ; 598(2): 220-232, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-37923554

RESUMO

Intracellular variable fragments of heavy-chain antibody from camelids (intra-VHH) have been successfully used as chaperones to solve the 3D structure of active G protein-coupled receptors bound to their transducers. However, their effect on signalling has been poorly explored, although they may provide a better understanding of the relationships between receptor conformation and activity. Here, we isolated and characterized iPRC1, the first intra-VHH recognizing a member of the large glycoprotein hormone receptor family, the follicle-stimulating hormone receptor (FSHR). This intra-VHH recognizes the FSHR third intracellular loop and decreases cAMP production in response to FSH, without altering Gαs recruitment. Hence, iPRC1 behaves as an allosteric modulator and provides a new tool to complete structure/activity studies performed thus far on this receptor.


Assuntos
Hormônio Foliculoestimulante , Receptores do FSH , Receptores do FSH/genética , Receptores do FSH/química , Receptores do FSH/metabolismo , Hormônio Foliculoestimulante/química , Hormônio Foliculoestimulante/metabolismo , Hormônio Foliculoestimulante/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Transdução de Sinais
4.
Int J Mol Sci ; 24(21)2023 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-37958944

RESUMO

Developing modulatory antibodies against G protein-coupled receptors is challenging. In this study, we targeted the follicle-stimulating hormone receptor (FSHR), a significant regulator of reproduction, with variable domains of heavy chain-only antibodies (VHHs). We built two immune VHH libraries and submitted them to multiplexed phage display approaches. We used next-generation sequencing to identify 34 clusters of specifically enriched sequences that were functionally assessed in a primary screen based on a cAMP response element (CRE)-dependent reporter gene assay. In this assay, 23 VHHs displayed negative or positive modulation of FSH-induced responses, suggesting a high success rate of the multiplexed strategy. We then focused on the largest cluster identified (i.e., PRC1) that displayed positive modulation of FSH action. We demonstrated that PRC1 specifically binds to the human FSHR and human FSHR/FSH complex while potentiating FSH-induced cAMP production and Gs recruitment. We conclude that the improved selection strategy reported here is effective for rapidly identifying functionally active VHHs and could be adapted to target other challenging membrane receptors. This study also led to the identification of PRC1, the first potential positive modulator VHH reported for the human FSHR.


Assuntos
Bacteriófagos , Receptores do FSH , Humanos , Receptores do FSH/genética , Receptores do FSH/metabolismo , Hormônio Foliculoestimulante/metabolismo , Transdução de Sinais , Sequenciamento de Nucleotídeos em Larga Escala , Bacteriófagos/genética
5.
Int J Mol Sci ; 24(15)2023 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-37569429

RESUMO

We demonstrate here that highly sensitive in vitro bioassays for FSH, TSH, and PTH can be set up in mouse Leydig Tumor Cells (mLTC), in addition to the normal LH/CG bioassay, after they were transfected with expression vectors encoding the corresponding Gs Protein-Coupled Receptors (GsPCR), such as FSHR, TSHR, or PTHR. Although the ß2 adrenergic receptor is also a GsPCR, its expression in mLTC led to a significant but very low cAMP response compared to those observed with FSH, TSH, or PTH. Similarly, after transfection of the GiPCR MT1 melatonin receptor, we did not observe any inhibitory effect by melatonin of the LH or hCG stimulation. Interestingly, after transfection of mLTC with the human kisspeptin receptor (hKpR), which is a GqPCR, we observed a dose-dependent synergy of 10-12-10-7 M kisspeptin variants with a fixed concentration of 0.3 nM LH or hCG. Without any exogenous receptor transfection, a 2 h preincubation with OT or AVP led to a dose-dependent cAMP response to a fixed dose of LH or hCG. Therefore, highly sensitive in vitro bioassays for various hormones and other GPCR ligands can be set up in mLTC to measure circulating concentrations in only 3-10 µL of blood or other body fluids. Nevertheless, the development of an LHRKO mLTC cell line will be mandatory to obtain strict specificity for these bioassays to eliminate potential cross-reaction with LH or CG.


Assuntos
Kisspeptinas , Receptores do LH , Camundongos , Animais , Humanos , Receptores do LH/genética , Receptores do LH/metabolismo , Kisspeptinas/metabolismo , Ligantes , AMP Cíclico/metabolismo , Transdução de Sinais , Receptores Acoplados a Proteínas G , Hormônio Foliculoestimulante/farmacologia , Hormônio Foliculoestimulante/metabolismo , Tireotropina/metabolismo , Gonadotropina Coriônica/metabolismo
6.
Front Endocrinol (Lausanne) ; 13: 1048601, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36465650

RESUMO

Single-domain antibody fragments, also known as VHHs or nanobodies, have opened promising avenues in therapeutics and in exploration of intracellular processes. Because of their unique structural properties, they can reach cryptic regions in their cognate antigen. Intracellular VHHs/antibodies primarily directed against cytosolic proteins or transcription factors have been described. In contrast, few of them target membrane proteins and even less recognize G protein-coupled receptors. These receptors are major therapeutic targets, which reflects their involvement in a plethora of physiological responses. Hence, they elicit a tremendous interest in the scientific community and in the industry. Comprehension of their pharmacology has been obscured by their conformational complexity, that has precluded deciphering their structural properties until the early 2010's. To that respect, intracellular VHHs have been instrumental in stabilizing G protein-coupled receptors in active conformations in order to solve their structure, possibly bound to their primary transducers, G proteins or ß-arrestins. In contrast, the modulatory properties of VHHs recognizing the intracellular regions of G protein-coupled receptors on the induced signaling network have been poorly studied. In this review, we will present the advances that the intracellular VHHs have permitted in the field of GPCR signaling and trafficking. We will also discuss the methodological hurdles that linger the discovery of modulatory intracellular VHHs directed against GPCRs, as well as the opportunities they open in drug discovery.


Assuntos
Anticorpos , Descoberta de Drogas , Monitorização Fisiológica , Proteínas de Membrana , Transdução de Sinais
7.
J Biol Chem ; 298(9): 102332, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35933010

RESUMO

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.


Assuntos
Receptor Tipo 1 de Hormônio Paratireóideo , AMP Cíclico/metabolismo , Heparina/metabolismo , Humanos , Ligantes , Conformação Proteica , Receptor Tipo 1 de Hormônio Paratireóideo/química , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Transdução de Sinais , beta-Arrestinas/metabolismo
8.
Methods Cell Biol ; 166: 179-203, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34752332

RESUMO

Arrestins are key proteins that serve as versatile scaffolds to control and mediate G protein coupled receptors (GPCR) activity. Arrestin control of GPCR functions involves their recruitment from the cytosol to plasma membrane-localized GPCRs and to endosomal compartments, where they mediate internalization, sorting and signaling of GPCRs. Several methods can be used to monitor trafficking of arrestins; however, live fluorescence imaging remains the method of choice to both assess arrestin recruitment to ligand-activated receptors and to monitor its dynamic subcellular localization. Here, we present two approaches based on Total Internal Fluorescence (TIRF) microscopy and confocal microscopy to visualize arrestin trafficking in live cells in real time and to assess their co-localization with the GPCR of interest and their localization at specific subcellular locations.


Assuntos
Arrestina , Arrestinas , Arrestinas/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestinas/metabolismo
9.
Sci Signal ; 14(703): eabc5944, 2021 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-34609896

RESUMO

The parathyroid hormone (PTH) type 1 receptor (PTHR) is a class B G protein­coupled 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 (PTH7d) that elicits cAMP production at the plasma membrane but not at endosomes. PTH7d stabilized a unique active PTHR conformation that mediated sustained cAMP signaling at the plasma membrane due to impaired ß-arrestin coupling to the receptor. Experiments in cells and mice revealed that sustained cAMP production by cell surface PTHR failed to mimic the pharmacological effects of sustained endosomal cAMP production on the abundance of the rate-limiting hydroxylase catalyzing the formation of active vitamin D, as well as increases in circulating active vitamin D and Ca2+ and in bone formation in mice. Thus, similar amounts of cAMP generated by PTHR for similar lengths of time in different cellular locations, plasma membrane and endosomes, mediate distinct physiological responses. These results unveil subcellular signaling location as a means to achieve specificity in PTHR-mediated biological outcomes and raise the prospect of rational drug design based upon spatiotemporal manipulation of GPCR signaling.


Assuntos
Hormônio Paratireóideo , Receptores de Hormônios Paratireóideos , AMP Cíclico
10.
Int J Mol Sci ; 22(18)2021 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-34576014

RESUMO

Follicle-stimulating hormone receptor (FSHR) plays a key role in reproduction through the activation of multiple signaling pathways. Low molecular weight (LMW) ligands composed of biased agonist properties are highly valuable tools to decipher complex signaling mechanisms as they allow selective activation of discrete signaling cascades. However, available LMW FSHR ligands have not been fully characterized yet. In this context, we explored the pharmacological diversity of three benzamide and two thiazolidinone derivatives compared to FSH. Concentration/activity curves were generated for Gαs, Gαq, Gαi, ß-arrestin 2 recruitment, and cAMP production, using BRET assays in living cells. ERK phosphorylation was analyzed by Western blotting, and CRE-dependent transcription was assessed using a luciferase reporter assay. All assays were done in either wild-type, Gαs or ß-arrestin 1/2 CRISPR knockout HEK293 cells. Bias factors were calculated for each pair of read-outs by using the operational model. Our results show that each ligand presented a discrete pharmacological efficacy compared to FSH, ranging from super-agonist for ß-arrestin 2 recruitment to pure Gαs bias. Interestingly, LMW ligands generated kinetic profiles distinct from FSH (i.e., faster, slower or transient, depending on the ligand) and correlated with CRE-dependent transcription. In addition, clear system biases were observed in cells depleted of either Gαs or ß-arrestin genes. Such LMW properties are useful pharmacological tools to better dissect the multiple signaling pathways activated by FSHR and assess their relative contributions at the cellular and physio-pathological levels.


Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/farmacologia , Receptores do FSH/agonistas , beta-Arrestina 2/farmacologia , AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , Cinética
11.
Nat Metab ; 2(3): 243-255, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32694772

RESUMO

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) B1 receptor (GABAB1R) in hyperplastic parathyroid glands (PTGs) of patients with primary and secondary HPT. Targeted ablation of GABAB1R 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. Cobinding of the CaSR/GABAB1R complex by baclofen and high extracellular calcium blocks the coupling of heterotrimeric G-proteins to homomeric CaSRs in cultured cells and promotes PTH secretion in cultured mouse PTGs. These results combined with the ability of PTG to synthesize GABA support a critical autocrine action of GABA/GABAB1R in mediating tonic PTH secretion of PTGs and ascribe aberrant activities of CaSR/GABAB1R heteromer to HPT.


Assuntos
Hiperparatireoidismo Secundário/metabolismo , Hormônio Paratireóideo/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Cálcio/metabolismo , Humanos , Hiperparatireoidismo Secundário/complicações , Hipocalcemia/complicações , Hipocalcemia/metabolismo , Camundongos , Receptores de GABA-B/metabolismo
12.
Nat Chem Biol ; 16(10): 1096-1104, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32632293

RESUMO

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.


Assuntos
Arrestina/metabolismo , Hormônio Paratireóideo/metabolismo , Arrestina/química , Fosfatos de Cálcio , Microscopia Crioeletrônica , AMP Cíclico , Escherichia coli , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Hormônio Paratireóideo/química , Receptores Acoplados a Proteínas G
13.
Proc Natl Acad Sci U S A ; 117(13): 7455-7460, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32184323

RESUMO

cAMP production upon activation of Gs 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 Gq/11 activation by the parathyroid hormone (PTH) type 1 receptor (PTHR) in mediating endosomal cAMP responses. Inhibition of Gq/11 signaling by FR900359 markedly reduced the duration of PTH-induced cAMP production, and this effect was mimicked in cells lacking endogenous Gαq/11 We determined that modulation of cAMP generation by Gq/11 occurs at the level of the heterotrimeric G protein via liberation of cell surface Gßγ subunits, which, in turn, act in a phosphoinositide-3 kinase-dependent manner to promote the assembly of PTHR-ßarrestin-Gßγ signaling complexes that mediate endosomal cAMP responses. These results unveil insights into the spatiotemporal regulation of Gs-dependent cAMP signaling.


Assuntos
AMP Cíclico/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Animais , Arrestinas/metabolismo , Membrana Celular/metabolismo , Depsipeptídeos/farmacologia , Endossomos/metabolismo , Células HEK293 , Humanos , Camundongos , Osteoblastos/metabolismo , Hormônio Paratireóideo/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Cultura Primária de Células , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , beta-Arrestinas/metabolismo
14.
J Am Chem Soc ; 141(37): 14486-14490, 2019 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-31496241

RESUMO

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.


Assuntos
Receptor Tipo 1 de Hormônio Paratireóideo/química , Sequência de Aminoácidos , AMP Cíclico/biossíntese , Células HEK293 , Humanos , Proteína Relacionada ao Hormônio Paratireóideo/farmacologia , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Homologia de Sequência de Aminoácidos , Transdução de Sinais
15.
Artigo em Inglês | MEDLINE | ID: mdl-30930853

RESUMO

Knowledge on G protein-coupled receptor (GPCRs) structure and mechanism of activation has profoundly evolved over the past years. The way drugs targeting this family of receptors are discovered and used has also changed. Ligands appear to bind a growing number of GPCRs in a competitive or allosteric manner to elicit balanced signaling or biased signaling (i.e., differential efficacy in activating or inhibiting selective signaling pathway(s) compared to the reference ligand). These novel concepts and developments transform our understanding of the follicle-stimulating hormone (FSH) receptor (FSHR) biology and the way it could be pharmacologically modulated in the future. The FSHR is expressed in somatic cells of the gonads and plays a major role in reproduction. When compared to classical GPCRs, the FSHR exhibits intrinsic peculiarities, such as a very large NH2-terminal extracellular domain that binds a naturally heterogeneous, large heterodimeric glycoprotein, namely FSH. Once activated, the FSHR couples to Gαs and, in some instances, to other Gα subunits. G protein-coupled receptor kinases and ß-arrestins are also recruited to this receptor and account for its desensitization, trafficking, and intracellular signaling. Different classes of pharmacological tools capable of biasing FSHR signaling have been reported and open promising prospects both in basic research and for therapeutic applications. Here we provide an updated review of the most salient peculiarities of FSHR signaling and its selective modulation.

16.
Science ; 364(6436): 148-153, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30975883

RESUMO

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. In contrast to a single TMD structure state, the extracellular domain adopts multiple conformations. These results provide insights into the structural basis and dynamics of PTH binding and receptor activation.


Assuntos
Hormônio Paratireóideo/química , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Receptor Tipo 1 de Hormônio Paratireóideo/química , Motivos de Aminoácidos , Microscopia Crioeletrônica , Humanos , Hormônio Paratireóideo/farmacologia , Hormônio Paratireóideo/fisiologia , Ligação Proteica , Domínios Proteicos , Receptor Tipo 1 de Hormônio Paratireóideo/ultraestrutura
17.
Proc Natl Acad Sci U S A ; 116(8): 3294-3299, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30718391

RESUMO

The parathyroid hormone (PTH) and its related peptide (PTHrP) activate PTH receptor (PTHR) signaling, but only the PTH sustains GS-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 Ca2+ acts as a positive allosteric modulator of PTHR signaling that regulates sustained cAMP production. Equilibrium and kinetic studies of ligand-binding and receptor activation reveal that Ca2+ prolongs the residence time of ligands on the receptor, thus, increasing both the duration of the receptor activation and the cAMP signaling. We further find that Ca2+ allostery in the PTHR is strongly affected by the point mutation recently identified in the PTH (PTHR25C) as a new cause of hypocalcemia in humans. Using high-resolution and mass accuracy mass spectrometry approaches, we identified acidic clusters in the receptor's first extracellular loop as key determinants for Ca2+ allosterism and endosomal cAMP signaling. These findings coupled to defective Ca2+ allostery and cAMP signaling in the PTHR by hypocalcemia-causing PTHR25C suggest that Ca2+ allostery in PTHR signaling may be involved in primary signaling processes regulating calcium homeostasis.


Assuntos
AMP Cíclico/genética , Hipocalcemia/genética , Hormônio Paratireóideo/genética , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Regulação Alostérica/genética , Animais , Células COS , Sinalização do Cálcio/genética , Chlorocebus aethiops , AMP Cíclico/metabolismo , Humanos , Hipocalcemia/metabolismo , Hipocalcemia/patologia , Cinética , Ligantes , Hormônio Paratireóideo/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/genética , Mutação Puntual/genética , Ligação Proteica/genética , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo
18.
J Biol Chem ; 294(12): 4546-4571, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30696771

RESUMO

Na+-H+ exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins, including sodium-phosphate co-transport protein 2A (NPT2A) at the plasma membrane. NHERF1 is a phosphoprotein with 40 Ser and Thr residues. Here, using tandem MS analysis, we characterized the sites of parathyroid hormone (PTH)-induced NHERF1 phosphorylation and identified 10 high-confidence phosphorylation sites. Ala replacement at Ser46, Ser162, Ser181, Ser269, Ser280, Ser291, Thr293, Ser299, and Ser302 did not affect phosphate uptake, but S290A substitution abolished PTH-dependent phosphate transport. Unexpectedly, Ser290 was rapidly dephosphorylated and rephosphorylated after PTH stimulation, and we found that protein phosphatase 1α (PP1α), which binds NHERF1 through a conserved VxF/W PP1 motif, dephosphorylates Ser290 Mutating 257VPF259 eliminated PP1 binding and blunted dephosphorylation. Tautomycetin blocked PP1 activity and abrogated PTH-sensitive phosphate transport. Using fluorescence lifetime imaging (FLIM), we observed that PTH paradoxically and transiently elevates intracellular phosphate. Added phosphate blocked PP1α-mediated Ser290 dephosphorylation of recombinant NHERF1. Hydrogen-deuterium exchange MS revealed that ß-sheets in NHERF1's PDZ2 domain display lower deuterium uptake than those in the structurally similar PDZ1, implying that PDZ1 is more cloistered. Dephosphorylated NHERF1 exhibited faster exchange at C-terminal residues suggesting that NHERF1 dephosphorylation precedes Ser290 rephosphorylation. Our results show that PP1α and NHERF1 form a holoenzyme and that a multiprotein kinase cascade involving G protein-coupled receptor kinase 6A controls the Ser290 phosphorylation status of NHERF1 and regulates PTH-sensitive, NPT2A-mediated phosphate uptake. These findings reveal how reversible phosphorylation modifies protein conformation and function and the biochemical mechanisms underlying PTH control of phosphate transport.


Assuntos
Hormônio Paratireóideo/fisiologia , Fosfatos/metabolismo , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/fisiologia , Sequência de Aminoácidos , Cristalografia por Raios X , Furanos/farmacologia , Células HEK293 , Humanos , Transporte de Íons/fisiologia , Lipídeos/farmacologia , Fosfoproteínas/química , Fosforilação , Conformação Proteica , Receptores de Neuropeptídeo Y/antagonistas & inibidores , Receptores de Neuropeptídeo Y/metabolismo , Serina/metabolismo , Trocadores de Sódio-Hidrogênio/química
19.
Cell Rep ; 21(10): 2855-2867, 2017 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-29212031

RESUMO

Endocytic trafficking is a critical mechanism for cells to decode complex signaling pathways, including those activated by G-protein-coupled receptors (GPCRs). Heterogeneity in the endosomal network enables GPCR activity to be spatially restricted between early endosomes (EEs) and the recently discovered endosomal compartment, the very early endosome (VEE). However, the molecular machinery driving GPCR activity from the VEE is unknown. Using luteinizing hormone receptor (LHR) as a prototype GPCR for this compartment, along with additional VEE-localized GPCRs, we identify a role for the adaptor protein APPL1 in rapid recycling and endosomal cAMP signaling without impacting the EE-localized ß2-adrenergic receptor. LHR recycling is driven by receptor-mediated Gαs/cAMP signaling from the VEE and PKA-dependent phosphorylation of APPL1 at serine 410. Receptor/Gαs endosomal signaling is localized to microdomains of heterogeneous VEE populations and regulated by APPL1 phosphorylation. Our study uncovers a highly integrated inter-endosomal communication system enabling cells to tightly regulate spatially encoded signaling.


Assuntos
Endossomos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , AMP Cíclico/metabolismo , Citometria de Fluxo , Células HEK293 , Humanos , Imunoprecipitação , Fosforilação , Transporte Proteico/fisiologia , Transdução de Sinais/fisiologia
20.
Physiol Rep ; 5(19)2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29038362

RESUMO

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). We found that FSS triggered an increase in intracellular Ca2+ concentration ([Ca2+]i) that required release of extracellular ATP and the presence of primary cilia. In this study, we elucidate steps downstream of the increase in [Ca2+]i that link FSS-induced calcium increase to increased apical endocytic capacity. Using an intramolecular FRET probe, we show that activation of Cdc42 is a necessary step in the FSS-stimulated apical endocytosis cascade. Cdc42 activation requires the primary cilia and the FSS-mediated increase in [Ca2+]i Moreover, Cdc42 activity and FSS-stimulated endocytosis are coordinately modulated by activators and inhibitors of calmodulin. Together, these data suggest a mechanism by which PT cell exposure to FSS is translated into enhanced endocytic uptake of filtered molecules.


Assuntos
Endocitose , Túbulos Renais Proximais/metabolismo , Estresse Mecânico , Proteína cdc42 de Ligação ao GTP/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Calmodulina/metabolismo , Linhagem Celular , Feminino , Túbulos Renais Proximais/citologia , Gambás , Transdução de Sinais
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