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

Recent work indicates that mitogen-activated protein kinase kinase (MEK)1 signaling at the G2/M cell cycle transition unlinks the contiguous mammalian Golgi apparatus and that this regulates cell cycle progression. Here, we sought to determine the role in this pathway of Golgi reassembly protein (GRASP)55, a Golgi-localized target of MEK/extracellular signal-regulated kinase (ERK) phosphorylation at mitosis. In support of the hypothesis that GRASP55 is inhibited in late G2 phase, causing unlinking of the Golgi ribbon, we found that HeLa cells depleted of GRASP55 show a fragmented Golgi similar to control cells arrested in G2 phase. In the absence of GRASP55, Golgi stack length is shortened but Golgi stacking, compartmentalization, and transport seem normal. Absence of GRASP55 was also sufficient to suppress the requirement for MEK1 in the G2/M transition, a requirement that we previously found depends on an intact Golgi ribbon. Furthermore, mimicking mitotic phosphorylation of GRASP55 by using aspartic acid substitutions is sufficient to unlink the Golgi apparatus in a gene replacement assay. Our results implicate MEK1/ERK regulation of GRASP55-mediated Golgi linking as a control point in cell cycle progression. INTRODUCTIONThe structural diversity of the Golgi apparatus among eukaryotes suggests two primary modes of organization. Cis-, medial-, and trans-Golgi cisternae, although unconnected in budding yeast, are typically present as membrane stacks. Whereas many cell types contain numerous scattered stacks positioned adjacent to endoplasmic reticulum (ER) exit sites, known as ministacks, mammalian cells and those from related metazoans exhibit a pericentrosomal Golgi in which ministacks are laterally linked into a ribbon. The in vivo requirements for Golgi stack formation remain unknown; however, recent work has begun to identify components necessary for linking cisternal stacks into a contiguous Golgi ribbon.Depletion of the golgin Golgi matrix protein 130 kDa (GM130), or its binding partner Golgi reassembly protein (GRASP)65, by using RNA interference (RNAi) specifically disrupts formation of the Golgi ribbon (Puthenveedu et al., 2006). GM130 seems to recruit GRASP65 to Golgi membranes because Golgi localization of GRASP65 is lost upon GM130 knockdown, whereas GM130 remains Golgi localized in the absence of GRASP65 (Sutterlin et al., 2005;Puthenveedu et al., 2006). A mechanism for Golgi ribbon formation by GRASP65 is suggested by the finding that two postsynaptic density 95/disc-large/zona occludens-like domains at the N terminus of GRASP65 form oligomers that, in the presence of cytosol, can cross-link beads (Wang et al., 2003(Wang et al., , 2005. Thus, the Golgi ribbon may be formed by GM130-dependent recruitment of GRASP65 to Golgi cisternal rims where GRASP65 transoligomer formation crossbridges adjacent cis-cisternae and possibly primes them for soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated membrane fusion (Puthenveedu et al., 2006).The lateral contacts that form the Gol...

Background: It remains unclear why vasopressin induces greater antidiuresis through V2R than does oxytocin. Results: Vasopressin sustains cAMP signaling during V2R internalization, a process promoted by ␤-arrestins, and is halted by the retromer complex. Conclusion: This new noncanonical model of GPCR signaling differentiates the actions of vasopressin and oxytocin.Significance: This emerging model may explain the physiological bias between ligands.

Two controversies have emerged regarding the signaling pathways that regulate Golgi disassembly at the G2/M cell cycle transition. The first controversy concerns the role of mitogen-activated protein kinase activator mitogen-activated protein kinase kinase (MEK)1, and the second controversy concerns the participation of Golgi structure in a novel cell cycle “checkpoint.” A potential simultaneous resolution is suggested by the hypothesis that MEK1 triggers Golgi unlinking in late G2 to control G2/M kinetics. Here, we show that inhibition of MEK1 by RNA interference or by using the MEK1/2-specific inhibitor U0126 delayed the passage of synchronized HeLa cells into M phase. The MEK1 requirement for normal mitotic entry was abrogated if Golgi proteins were dispersed before M phase by treatment of cells with brefeldin A or if GRASP65, which links Golgi stacks into a ribbon network, was depleted. Imaging revealed that unlinking of the Golgi apparatus begins before M phase, is independent of cyclin-dependent kinase 1 activation, and requires MEK signaling. Furthermore, expression of the GRASP family member GRASP55 after alanine substitution of its MEK1-dependent mitotic phosphorylation sites inhibited both late G2 Golgi unlinking and the G2/M transition. Thus, MEK1 plays an in vivo role in Golgi reorganization, which regulates cell cycle progression.

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.