Robert A. Nissenson

Parathyroid hormonelike protein from human renal carcinoma cells. Structural and functional homology with parathyroid hormone.

A variety of solid tumors secrete proteins that are immunochemically distinct from parathyroid hormone (PTH) but activate PTH-responsive adenylate cyclase. Such PTH-like proteins have been proposed as mediators of the hypercalcemia and hypophosphatemia frequently associated with malignancies. We purified to apparent homogeneity a PTH-like protein with a molecular weight of 6,000, that is produced by human renal carcinoma cells. The amino-terminal sequence of the PTH-like protein and that of human PTH were found to display at least five identities in the first 13 positions. The purified protein bound to PTH receptors, activated adenylate cyclase in renal plasma membranes, and stimulated cAMP formation in rat osteosarcoma cells. The PTH-like protein reproduced two additional effects of PTH, stimulation of bone resorption in fetal rat limb bone cultures and inhibition of phosphate uptake in cultured opossum kidney cells. These properties are consistent with a role for PTH-like proteins as mediators of the syndrome of malignancy-associated hypercalcemia.

PTH Differentially Regulates Expression of RANKL and OPG

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.

Increased Serum Levels of a Parathyroid Hormone-like Protein in Malignancy-Associated Hypercalcemia

STUDY OBJECTIVE To measure the serum levels of a newly described parathyroid hormone-like protein (PLP) which was isolated from malignant tumors associated with hypercalcemia, and determine whether PLP is a humoral factor in malignancy-associated hypercalcemia. DESIGN A cross-sectional study of serum levels of PLP using a newly developed radioimmunoassay. SETTING A university-affiliated Veterans Administration hospital in San Francisco, California, a University hospital in Hong Kong, and a private hospital in Danville, Pennsylvania. PATIENTS Patients with hypercalcemia (calcium greater than 2.65 mmol/L) and a diagnosis of malignancy were studied. Control groups included normocalcemic patients with malignancy, patients with hyperparathyroidism, and normal subjects. MEASUREMENTS AND MAIN RESULTS Serum immunoreactive PLP (iPLP) levels in normal subjects were less than 2.5 pmol eq/L (10 pg/mL), and 68% of subjects had undetectable levels. The serum concentration of iPLP was normal in 15 of 16 hypercalcemic patients with hyperparathyroidism. Serum iPLP was increased (greater than 2.5 pmol eq/L) in 36 of 65 (55%) patients with malignancy-associated hypercalcemia, with a mean value of 6.1 +/- 0.9 pmol eq/L (24 pg/mL). In a subgroup of patients with solid tumors serum iPLP was increased in 30 (71%) of 42 hypercalcemic patients, with a mean value of 6.5 +/- 0.9 pmol eq/L. Serum iPLP was elevated in only 3 of 23 normocalcemic patients with cancer. In patients with solid malignancies (n = 59), levels of iPLP were positively correlated with the total serum calcium (r = 0.43, P less than 0.01). CONCLUSION The data indicate a relation between the serum concentration of iPLP and the presence of hypercalcemia in solid malignancies. The results support a role for PLP as a humoral mediator of hypercalcemia in most patients with solid tumors. Measurement of iPLP should be useful in the differential diagnosis of hypercalcemia.

Similar Structures and Shared Switch Mechanisms of the β2-Adrenoceptor and the Parathyroid Hormone Receptor Zn(II) BRIDGES BETWEEN HELICES III AND VI BLOCK ACTIVATION

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, Gt (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 β2-adrenoreceptor and the parathyroid hormone receptor; occupancy of the metal-binding site by Zn(II) markedly impairs the ability of each receptor to mediate ligand-dependent activation of Gs, 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.

Engineering GPCR signaling pathways with RASSLs

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 and Gq). We review these advances here to facilitate the use of these powerful and diverse tools.

Human renal carcinoma cells produce hypercalcemia in the nude mouse and a novel protein recognized by parathyroid hormone receptors.

When grown in nude mice, cultured renal carcinoma cells from a hypercalcemic patient produced marked hypercalcemia that was reversed by resection of tumor. Conditioned medium from this cell line contained a protein with activity in a renal adenylate cyclase bioassay for parathyroid hormone (PTH) which was blocked by the competitive PTH antagonist [8norleucyl, 18norleucyl, 34tyrosinyl]bPTH (3-34)amide. However, the biologically active protein was eluted from gel filtration columns as a larger molecular size component that PTH and was not recognized by any of four region-specific PTH antisera. The properties of this factor resemble those of the postulated PTH-like substance(s) in humoral hypercalcemia of malignancy.

The N-terminal Region of the Third Intracellular Loop of the Parathyroid Hormone (PTH)/PTH-related Peptide Receptor Is Critical for Coupling to cAMP and Inositol Phosphate/Ca2+ Signal Transduction Pathways

Structural determinants within the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor that mediate G-protein activation of adenylate cyclase and phospholipase C are unknown. We investigated the role of the N-terminal region of the third intracellular loop of the opossum PTH/PTHrP receptor in coupling to two signal transduction pathways. We mutated residues in this region by tandem-alanine scanning and expressed these mutant receptors in COS-7 cells and/or Xenopus oocytes. All mutant receptors retained high affinity PTH binding in COS-7 cells, indistinguishable from wild-type receptors. Receptors with tandem-alanine substitutions in two N-terminal segments (377RVL379 and 381TKLR384) demonstrated impaired adenylate cyclase and phospholipase C activation. Receptor mutants with single-alanine substitutions scanning these two segments showed three different signaling defects in COS-7 cells. 1) Two mutant receptors (V378A and L379A) had reduced inositol phosphate (IP), but normal cAMP responses to PTH. 2) Mutant receptor T381A showed reduced cAMP, but wild-type IP responses to PTH. 3) Mutant receptor K382A demonstrated both markedly reduced cAMP and IP production due to PTH. In oocytes, mutants T381A and K382A showed decreased PTH-stimulated cAMP accumulation and intracellular Ca2+ mobilization. Thus, the N-terminal region of the third intracellular loop of this receptor plays a critical role in coupling to both Gs- and Gq-mediated second-messenger generation.

Osteoblast expression of an engineered Gs-coupled receptor dramatically increases bone mass

Osteoblasts are essential for maintaining bone mass, avoiding osteoporosis, and repairing injured bone. Activation of osteoblast G protein-coupled receptors (GPCRs), such as the parathyroid hormone receptor, can increase bone mass; however, the anabolic mechanisms are poorly understood. Here we use “Rs1,” an engineered GPCR with constitutive Gs signaling, to evaluate the temporal and skeletal effects of Gs signaling in murine osteoblasts. In vivo, Rs1 expression induces a dramatic anabolic skeletal response, with midfemur girth increasing 1,200% and femur mass increasing 380% in 9-week-old mice. Bone volume, cellularity, areal bone mineral density, osteoblast gene markers, and serum bone turnover markers were also elevated. No such phenotype developed when Rs1 was expressed after the first 4 weeks of postnatal life, indicating an exquisite temporal sensitivity of osteoblasts to Rs1 expression. This pathway may represent an important determinant of bone mass and may open future avenues for enhancing bone repair and treating metabolic bone diseases.

Resolution Dependence of the Non-metric Trabecular Structure Indices

Non-metric indices of topological features of trabecular bone structure, such as structure model index (SMI), connectivity density (Conn.D), and degree of anisotropy (DA), provide unique information relevant to bone quality. With recent technological advancement, in vivo assessment of these indices may be possible from images acquired using high-resolution imaging techniques such as high-resolution peripheral quantitative computed tomography (HR-pQCT). However, more detailed investigation of the dependence of non-metric indices on spatial resolution is needed to determine their applicability. The purpose of this study was to determine whether these three non-metric indices are affected by the spatial resolution of CT images. First, the SMI, Conn.D, and DA were calculated for trabecular bone specimens with varying plate-like and rod-like structures from resampled muCT images across a range of spatial resolutions and compared to the reference values. To account for differences in size across different species and anatomical sites, the results are reported in normalized resolution units. Next, the impact of resolution on the non-metric indices for cores of human distal tibia trabecular bone from clinical HR-pQCT images was evaluated to determine the applicability of the non-metric indices to in vivo imaging. We found that the non-metric indices of trabecular bone structure were affected by spatial resolution of CT images. Particularly, the SMI deviated from the high-resolution muCT reference value depending on the structure type, whether plate-like or rod-like. Both Conn.D and DA were underestimated in the images obtained at an in vivo resolution. It is not trivial to determine absolute threshold for validity of these non-metric indices without considering a specific study design (e.g. relative resolution, the size of the treatment effect to detect, and specimen type). The results of this study provide an upper bound for the accuracy of the non-metric indices under limited resolution scenarios.

Mitogenic Gi protein‐MAP kinase signaling cascade in MC3T3‐E1 osteogenic cells: Activation by C‐terminal pentapeptide of osteogenic growth peptide [OGP(10–14)] and attenuation of activation by cAMP

In osteogenic and other cells the mitogen‐activated protein (MAP) kinases have a key role in regulating proliferation and differentiated functions. The osteogenic growth peptide (OGP) is a 14 mer mitogen of osteogenic and fibroblastic cells that regulates bone turnover, fracture healing, and hematopoiesis, including the engraftment of bone marrow transplants. It is present in the serum and extracellular fluid either free or complexed to OGP‐binding proteins (OGPBPs). The free immunoreactive OGP consists of the full length peptide and its C‐terminal pentapeptide OGP(10–14). In the present study, designed to probe the signaling pathways triggered by OGP, we demonstrate in osteogenic MC3T3 E1 cells that mitogenic doses of OGP(10–14), but not OGP, enhance MAP kinase activity in a time‐dependent manner. The OGP(10–14)‐induced stimulation of both MAP kinase activity and DNA synthesis were abrogated by pertusis toxin, a Gi protein inhibitor. These data offer direct evidence for the occurrence in osteogenic cells of a peptide‐activated, mitogenic Gi protein‐MAP kinase‐signaling cascade. Forskolin and dBu2‐cAMP abrogated the OGP(10–14)‐stimulated proliferation, but induced only 50% inhibition of the OGP(10–14)‐mediated MAP kinase activation, suggesting additional MAP kinase‐dependent, OGP(10–14)‐regulated, cellular functions. Finally, it is demonstrated that OGP(10–14) is the active form of OGP, apparently generated proteolytically in the extracellular milieu upon dissociation of OGP–OGPBP complexes. J. Cell. Biochem. 81: 594–603, 2001.