Studying the regulation of endosomal cAMP production in GPCR signaling

The parathyroid hormone receptor-1 (PTHR1) is a member of the B-family of GPCRs; these receptors are activated by long polypeptide hormones and constitute targets of drug development efforts. Parathyroid hormone (PTH; 84 residues) and PTH-related protein (PTHrP; 141 residues) are natural agonists of PTHR1, and an N-terminal fragment of PTH, PTH(1−34), is used clinically to treat osteoporosis. Conventional peptides in the 20-40-mer length range are rapidly degraded by proteases, which may limit their biomedical utility. We have used the PTHR1-ligand system to explore the impact of broadly distributed replacement of α-amino acid residues with β-amino acid residues on susceptibility to proteolysis and agonist activity. This effort led us to identify new PTHR1 agonists that contain α→β replacements throughout their sequences, manifest potent agonist activity in cellular assays, and display remarkable resistance to proteolysis. The strategy we have employed suggests a path toward identifying protease-resistant agonists of other B-family GPCRs.

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“…57, 63, 67–69 The mechanistic underpinnings of this correlation are an active area of investigation. 64, 66, 70–74 …”

Section: Resultsmentioning

confidence: 99%

Development of Potent, Protease-Resistant Agonists of the Parathyroid Hormone Receptor with Broad β Residue Distribution

et al. 2017

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“…Peptides and Chemicals. PTH , the N-terminal synthetic analog of naturally circulating PTH(1−84), and TMR-labeled PTH(1-34) were generated as previously described (25). Forskolin (#344270) was purchased from EMD-Millipore.…”

Section: Methodsmentioning

confidence: 99%

“…Cells were transiently transfected with the FRET-based biosensor, Epac1 CFP/YFP (29), for measuring cAMP. Measurements were performed and analyzed as previously described (25). In brief, cells plated on poly-D-lysine coated glass coverslips were mounted in Attofluor cell chambers (Life Technologies), maintained in Hepes buffer containing 150 mM NaCl, 20 mM Hepes, 2.5 mM KCl, 1 mM CaCl 2 , and 0.1% BSA at pH 7.4, and cell chambers were placed on the oil immersion 40× N.A 1.30 Plan Apo objective of our Nikon Ti-E microscope (Nikon Corporation).…”

Section: Methodsmentioning

confidence: 99%

G _q/11 -dependent regulation of endosomal cAMP generation by parathyroid hormone class B GPCR

White

Jean-Alphonse

Fang

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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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 Gβγ subunits, which, in turn, act in a phosphoinositide-3 kinase-dependent manner to promote the assembly of PTHR–βarrestin–Gβγ signaling complexes that mediate endosomal cAMP responses. These results unveil insights into the spatiotemporal regulation of Gs-dependent cAMP signaling.

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“…The HA peptides were removed by using an Amicon Ultra 0.5 ml centrifuge filter (MWCO, 10 kDa, Sigma #Z677108). The endosomal PTHR signaling complexes were digested using an in-solution digestion protocol as described previously 32–34 . Tryptic peptides were subjected to LC–MS/MS analyses.…”

Section: Methodsmentioning

confidence: 99%

β2-adrenergic receptor control of endosomal PTH receptor signaling via Gβγ

et al. 2016

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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 β2-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.

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Studying the regulation of endosomal cAMP production in GPCR signaling

Cited by 8 publications

References 17 publications

Development of Potent, Protease-Resistant Agonists of the Parathyroid Hormone Receptor with Broad β Residue Distribution

Development of Potent, Protease-Resistant Agonists of the Parathyroid Hormone Receptor with Broad β Residue Distribution

G _q/11 -dependent regulation of endosomal cAMP generation by parathyroid hormone class B GPCR

β2-adrenergic receptor control of endosomal PTH receptor signaling via Gβγ

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Studying the regulation of endosomal cAMP production in GPCR signaling

Cited by 8 publications

References 17 publications

Development of Potent, Protease-Resistant Agonists of the Parathyroid Hormone Receptor with Broad β Residue Distribution

Development of Potent, Protease-Resistant Agonists of the Parathyroid Hormone Receptor with Broad β Residue Distribution

G q/11 -dependent regulation of endosomal cAMP generation by parathyroid hormone class B GPCR

β2-adrenergic receptor control of endosomal PTH receptor signaling via Gβγ

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G _q/11 -dependent regulation of endosomal cAMP generation by parathyroid hormone class B GPCR