Retromer Terminates the Generation of cAMP by Internalized PTH Receptors

Abstract

Generation of cAMP by G protein-coupled receptors (GPCRs) and its termination is currently thought to occur exclusively at the plasma membrane of cells. Under existing models of receptor regulation, this signal is primarily restricted by desensitizationof the receptors through their binding to β-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 β-arrestins are known to rapidly bind and internalize activated PTHR. Here we show that β-arrestin1 binding prolongs rather than terminates cAMP generation by PTHR, and that cAMP generation correlates with the persistence of arrestin-receptor complexes on endosomes. We found that PTHR signaling is instead turned-off by the retromer complex, which regulates traffic of internalized receptor from endosomes to the Golgi apparatus. Thus, binding by the retromer complex regulates sustained cAMP generation triggered by an internalized GPCR.The production of cAMP by activated GPCRs is traditionally thought to originate exclusively at the plasma membrane. In most receptors studied to date elevated cAMP is rapidly extinguished by mechanisms that desensitized activated receptors through phosphorylation by G protein receptor kinases, arrestin binding that prevents further G protein coupling and can recruit cAMP specific phosphodiesterases (PDE4D) to the plasma membrane 1 , and receptor endocytosis 2 , after which desensitized receptors are recycled to the Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms ¶ Corresponding author: Jean-Pierre Vilardaga, Ph. D., Department of Pharmacology and Chemical Biology, E1357 Thomas E. Starzl Biochemical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15261, Tel: (412) 648 2055, Fax: (412) 648 1945, jpv@pitt.edu. Note: Supplementary information is available on the Nature Chemical Biology website. Competing interests statementThe authors have no competing financial interests to disclose. Author contributionsT.N.F performed most of the experiments with the support of V.L.W., J.A., D.S.W., S.F., and T.J.G.; J.-P.V. designed and supervised the experiments; J.-P.V. and T.N.F. analyzed the data and wrote the manuscript; all authors discussed the results and commented on the manuscript. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author Manuscript plasma membrane or trafficked to lysosomes for degradation 3 . In either case, production of cAMP is not thought to continue after GPCR internalization. However, recent studies have shown that cAMP production mediated by the PTHR in response to PTH or PTH analogs continues even after internalization of the activated receptor 4 , 5 . Together with recent work on the thyroid-stimulating hormone receptor 6 , 7 , these observati...

Noncanonical GPCR signaling arising from a PTH receptor–arrestin–Gβγ complex

“…S1). Importantly, our previous studies showed that cAMP production induced by M-PTH(1-28) remains elevated for at least 30 min after ligand challenge (14).…”

“…For these assays, Gβγ CFP was expressed in HEK293 cells stably expressing HA-tagged PTHR (HA-PTHR), and cell lysates were prepared at different time points after a short exposure to either M-PTH(1-28) or M-PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), modified analogs of PTH that induce longer or shorter cAMP generation and biological responses, respectively (20). The difference in duration of cAMP signaling is thought to depend on the capacity of M-PTH(1-28), like PTH , to form an unusually persistent high-affinity complex with PTHR that is independent of G-protein coupling, whereas M-PTH(1-14) forms a more conventional high-affinity complex that is transient and dependent on G-protein coupling (2,20,21).…”

“…The difference in duration of cAMP signaling is thought to depend on the capacity of M-PTH(1-28), like PTH , to form an unusually persistent high-affinity complex with PTHR that is independent of G-protein coupling, whereas M-PTH(1-14) forms a more conventional high-affinity complex that is transient and dependent on G-protein coupling (2,20,21). The interactions of HA-PTHR with Gβγ CFP and β-arrestins, weakly detectable under basal conditions, increased significantly within 5 min after challenge with M-PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) or M-PTH(1-28) (Fig. S1).…”

“…We therefore tested whether stabilizing arrestin association with PTHR would also permit its interaction with Gβγ. To do this, we artificially immobilized an N-terminally GFP-tagged PTHR (PTHR-GFP) (22) at the plasma membrane of HEK293 cells and used a mutant of β-arrestin 1, I 386 A, V 387 A (hereafter β-arr1[IV-AA], or β-arr1[IV-AA] Tom when tagged with tdTomato), that exhibits increased binding affinity for ligand-activated PTHR (14). Cell-surface cross-linking effectively immobilized PTHR without affecting its signaling capacity (Fig.…”

“…We recently showed that parathyroid hormone receptor type 1 (PTHR)-arrestin complexes contribute to prolonging cAMP signaling mediated by PTH or its fully functional N-terminal synthetic analog, PTH , in cells as diverse as human embryonic kidney (HEK)293 cells expressing recombinant receptor and osteoblastic ROS17/2.8 cells (2,14) that natively express PTHR. PTH rapidly recruits β-arrestin 1 or β-arrestin 2 to PTHR and stabilizes a persistent ternary PTH-PTHR-arrestin complex that continues signaling via adenylyl cyclases for a considerable time (>30 min) after exposure to a short "pulse" of agonist ligand (14). Because prolonged PTH-induced signaling requires persistent activation of G S by coupling with PTHR in its L-R* state, this raises the intriguing question of how G S can bind and be activated by a PTHR that is already associated with arrestin.…”