RANKL and OPG gene expressions were measured with and without PTH at different stages of osteoblast development. Mouse stromal cells were cultured in osteoblast differentiating conditions, and RANKL, OPG, COL1, ALP, OC, and PTHRec genes were measured using qRT-PCR. OPG:RANKL ratios indicate that PTH may induce a possible switch in the regulatory mechanism of osteoclastogenesis where OPG is inhibited early and RANKL is increased at late stages of osteoblast differentiation. Introduction: RANKL is essential for osteoclastogenesis, and its decoy receptor osteoprotegerin (OPG) negatively regulates this process. Both genes are expressed in cells of the osteoblast lineage, but the precise relationship between the state of osteoblast differentiation and RANKL and OPG expression is not clearly defined. The goal of this project was to quantify changes in RANKL and OPG gene expression in response to parathyroid hormone (PTH) at different stages of osteoblast differentiation. In this study, mouse primary bone marrow stromal cells (BMSCs) were cultured for up to 28 days. At specific time-points of cell culture, cells were stimulated with bovine PTH peptide [bPTH (1-34)] for 2 h. Levels of RANKL, OPG, ␣-1 (type I) collagen (COL1), alkaline phosphatase (ALP), osteocalcin (OC), and PTH receptor (PTHRec) mRNA were assayed using quantitative real-time reverse-transcriptasepolymerase chain reaction (qRT-PCR). Materials and Methods: In control cells, there was a gradual increase of RANKL gene expression with murine osteoblastic stromal cell maturation to a 3-fold level at day 28. In contrast, OPG mRNA levels were maximal at day 14 of cell culture and decreased through the latter stages of osteoblast differentiation. Exposing the cells to 100 ng/ml of bPTH(1-34) induced minimal increases in RANKL mRNA levels from days 7 to 14 but elevated expression significantly at days 21 (2-fold) and 28 (3-fold). PTH inhibited OPG gene expression maximally at day 14, but continued to have inhibitory effects on cultured cells at days 21 and 28. Alterations of RANKL and OPG mRNA levels by PTH in day 14 osteoblasts were sufficient to sustain a 5.6-fold increase in the number of TRACP ϩ cells when cocultured with osteoclast precursor cells. Cells in culture after 28 days showed a 1.9-fold increase in TRACP

We are creating families of designer G-protein-coupled receptors (GPCRs) to allow for precise spatiotemporal control of GPCR signaling in vivo. These engineered GPCRs, called receptors activated solely by synthetic ligands (RASSLs), are unresponsive to endogenous ligands but can be activated by nanomolar concentrations of pharmacologically inert, drug-like small molecules. Currently, RASSLs exist for the three major GPCR signaling pathways (Gs, Gi, Gq). These new advances are reviewed here to help facilitate the use of these powerful and diverse tools.

The seven transmembrane helices of serpentine receptors comprise a conserved switch that relays signals from extracellular stimuli to heterotrimeric G proteins on the cytoplasmic face of the membrane. By substituting histidines for residues at the cytoplasmic ends of helices III and VI in retinal rhodopsin, we engineered a metal-binding site whose occupancy by Zn(II) prevented the receptor from activating a retinal G protein, G t (Sheikh, S. P., Zvyaga, T. A., Lichtarge, O., Sakmar, T. P., and Bourne, H. R. (1996) Nature 383, 347-350). Now we report engineering of metal-binding sites bridging the cytoplasmic ends of these two helices in two other serpentine receptors, the ␤ 2 -adrenoreceptor and the parathyroid hormone receptor; occupancy of the metal-binding site by Zn(II) markedly impairs the ability of each receptor to mediate liganddependent activation of G s , the stimulatory regulator of adenylyl cyclase. We infer that these two receptors share with rhodopsin a common three-dimensional architecture and an activation switch that requires movement, relative to one another, of helices III and VI; these inferences are surprising in the case of the parathyroid hormone receptor, a receptor that contains seven stretches of hydrophobic sequence but whose amino acid sequence otherwise shows no apparent similarity to those of receptors in the rhodopsin family. These findings highlight the evolutionary conservation of the switch mechanism of serpentine receptors and help to constrain models of how the switch works.Serpentine receptors are key signaling molecules that relay extracellular signals from hormones and sensory stimuli to heterotrimeric G proteins located on the cytoplasmic face of the plasma membrane. Ligand-activated receptors activate G proteins by promoting exchange of GTP for GDP bound to the ␣ subunit (G␣) of the heterotrimer, causing liberation of both ␣-GTP and free ␤␥ complexes, which in turn activate effector enzymes and ion channels (1, 2). Patterns of conserved amino acid sequence distinguish three separate families of serpentine receptors in mammals; these include the rhodopsin-like receptors, with more than 1000 members, and two smaller families, related to the secretin receptor or to metabotropic glutamate receptors, respectively (3-6). Although the three families share no similarities of primary structure, all members of each family activate heterotrimeric G proteins, and all contain seven stretches of hydrophobic amino acids, which are thought to be folded into a bundle of transmembrane ␣-helices.Baldwin et al. (7) have proposed a three-dimensional model of the transmembrane helices of receptors in the rhodopsin family. Based on analysis of the amino acid sequences of ϳ500 rhodopsin-like receptors and guided by a projection density map of frog rhodopsin (8), the model places each individual helix in the density map and specifies its position relative to the lipid bilayer, tilt in the plane of the membrane, and position and orientation relative to other helices. Thus experiments that define d...