Sustained cyclic AMP production by parathyroid hormone receptor endocytosis

Abstract

Cell signaling mediated by the G protein-coupled parathyroid hormone receptor type 1 (PTHR) is fundamental to bone and kidney physiology. It has been unclear how the two ligand systems-PTH, endocrine and homeostatic, and PTH-related peptide (PTHrP), paracrine-can effectively operate with only one receptor and trigger different durations of the cAMP responses. Here we analyze the ligand response by measuring the kinetics of activation and deactivation for each individual reaction step along the PTHR signaling cascade. We found that during the time frame of G protein coupling and cAMP production, PTHrP 1-36 action was restricted to the cell surface, whereas PTH 1-34 had moved to internalized compartments where it remained associated with the PTHR and Gα s , potentially as a persistent and active ternary complex. Such marked differences suggest a mechanism by which PTH and PTHrP induce differential responses, and these results indicate that the central tenet that cAMP production originates exclusively at the cell membrane must be revised.Seminal studies during the past decades established that signaling cascades mediated by a G protein-coupled receptor (GPCR) initially proceed through a succession of biochemical events that take place at the cell membrane and result in the induction and propagation of second messenger molecules 1-4 (Fig. 1a). These events begin with the binding of an 'agonist' ligand (L) to an inactive-state receptor (R), which causes the receptor to switch to an active-state conformation (R*). The activated receptor then interacts with heterotrimeric G proteins (G, or Gαβγ) to form a transient L-R*-G complex, which exhibits higher affinity for the agonist ligand than does the initial L-R state. The interaction process further involves a conformational change-induced exchange of GDP for GTP on Gα with concomitant release of the activated, GTP-bound Gα (along with Gβγ) from the L-R complex, and the

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

“…S3A). When PTHR was activated by M-PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), the diffusion of Gβγ remained unchanged (Fig. S3B).…”

“…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).…”

“…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 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.…”

“…Recent findings have challenged this view. 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).…”

“…A ccording to the current model for activation and signaling of G protein-coupled receptors (GPCRs), receptor (R) signaling begins when the binding of an agonist ligand (L) stabilizes the active form of the receptor (R*), allowing its coupling to heterotrimeric G proteins (Gαβγ) through a diffusion-controlled process (1)(2)(3). The L-R*-G complex catalyzes GDP-GTP exchange on the Gα subunit, promoting the dissociation of the activated G protein into GTP-bound Gα (Gα-GTP) and Gβγ dimers from the receptor.…”