Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-07-31
2001-02-06
Ulm, John (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C007S158000, C435S091500, C435S091500
Reexamination Certificate
active
06183974
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to G protein coupled receptors. More specifically, screening assays for Gs and Gq protein coupled receptor agonists and antagonists are provided. Also provided are stably transfected cell lines.
2. Related Art
Parathyroid hormone (PTH) is a major systemic regulator of bone turnover, and the closely related peptide, PTHrP, which is widely expressed in adult and fetal tissues, is believed to exert important paracrine actions, especially in developing cartilage and bone (Dempster, D. W., et al.,
Endocrine Rev
14:690-709 (1993); Lanske, B., et al.,
Science
273:663-666 (1996); Lee, K., et al.,
Endocrinology
137:5109-5118 (1996); Rixon, R. H., et al.,
J Bone Miner Res
9:1179-1189 (1994)). Exogenously administered PTH exerts striking effects upon bone mass in vivo, the nature of which depends critically upon the dose of PTH and the resulting temporal profile of the concentration of circulating peptide (Dempster, D. W., et al.,
Endocrine Rev
14:690-709 (1993)). Thus, continuous exposure to high PTH concentrations leads to net bone resorption and osteopenia, whereas intermittent administration of low doses leads to increased net bone formation—a finding that has fueled great interest in the possible use of PTH, or PTH analogs, as anabolic agents to increase bone mass and to prevent or treat metabolic bone diseases, including osteoporosis (Dempster, D. W., et al.,
Endocrine Rev
14:690-709 (1993), Whitfield, J. F., and Morley, P.,
Trends Pharmacol Sci
16:382-386 (1995)).
Both PTH and PTHrP can activate a single receptor, the PTH/PTHrP receptor (PTHR), which has been cloned from several species, including rat, opossum, mouse, pig and human, and shown to be expressed in cells of bone (Abou-Samra, A. B., et al.,
Proc Natl Acad Sci USA
89:2732-2736 (1992); Juppner, H., et al.,
Science
254:1024-1026 (1991); Schneider, H.,
Eur J Pharmacol
246:149-155 (1993); Bringhurst, F. R., et al.,
Endocrinology
132:2090-2098 (1991); Pines, M., et al.,
Endocrinology
135:1713-1716 (1994)). Activation of the PTHR in osteoblasts evokes multiple parallel signaling events, including activation of adenylyl cyclase (AC), phospholipase C (PLC) and cytosolic free calcium transients (Abou-Samra, A. B., et al.,
Proc Natl Acad Sci USA
89:2732-2736 (1992), Juppner, H., et al.,
Science
254:1024-1026 (1991), Bringhurst, F. R., et al.,
Endocrinology
132:2090-2098 (1993), Dunlay, R., and Hruska, K.,
Am J Physiol
258:F223-231 (1990); Fujimori, A., et al.,
Endocrinology
128:3032-3039 (1991); Yamaguchi, D. T., et al.,
J Biol Chem
262:7711-7718 (1987)). The links between each of these individual signaling events and the ultimate integrated tissue responses to PTH, such as changes in overall bone mass, remain largely undefined. It has been reported that PTH analogs which appear to selectively activate AC can generate a full anabolic effect on bone following intermittent administration in vivo (Rixon, R. H., et al.,
J Bone Miner Res
9:1179-1189 (1994), Whitfield, J. F., and Morley, P.,
Trends Pharmacol Sci
16:382-386 (1995), Whitfield, J. F., et al.,
Calcif Tissue Int
58:81-87 (1996)). Such observations have suggested that individual PTH second messengers may indeed be linked to specific tissue responses and, therefore, that the pattern of PTHR signaling events, as well as their intensity, may dictate both the qualitative and quantitative aspects of the response in bone. The issue is complicated by the fact that, in bone, mature osteoclasts are believed not to express PTHRs and thus must experience these influences of PTH only indirectly via responses generated by adjacent cells, such as osteoblasts or marrow stromal cells, which do express these receptors (Dempster, D. W., et al.,
Endocrine Rev
14:690-709 (1993), McSheehy, P., and Chambers, T.,
Endocrinology
118:824-828 (1986)). The manner whereby such osteoblastic or stromal PTH target cells might convey, to neighboring cells of the osteoclastic lineage, complex information reflecting subtle differences in temporal and concentration profiles of PTH exposure remains obscure.
Striking desensitization and downregulation of PTHRs has been described following exposure to high concentrations of ligand (Fujimori, A., et al.,
Endocrinology
128:3032-3039 (1991), Abou-Samra, A-B, et al.,
Endocrinology
129:2547-2554 (1991); Mitchell, J., and Goltzman, D.,
Endocrinology
126:2650-2660 (1990); Fukayama, S., et al.,
Endocrinology
131:1757-1769 (1992)) and it was reported recently that the pattern of signaling events generated by the rat PTHR is strongly influenced by the level of its expression on the cell surface (Guo, J., et al.,
Endocrinology
136:3884-3891 (1995)). Specifically, it was found that the magnitude of the PLC response was directly related to the density of available PTHRs on the surface of stably transfected LLC-PK1 cells across a range of expression (40,000-300,000 receptors per cell) that did not affect the maximal AC response (Guo, J., et al.,
Endocrinology
136:3884-3891 (1995)).
The human PTHR has been expressed previously in cultured cells (Pines, M., et al.,
Endocrinology
135:1713-1716 (1994), Schneider, H., et al.,
FEBS Lett
351:281-285 (1994), Pines, M., et al.,
Bone
18:381-389 (1996)), but its signaling properties have not yet been elucidated fully. In particular, the effects of alterations in human PTHR expression on the character of the signal transduction response(s) have not been systematically analyzed. Certain amino- or carboxyl-terminally truncated PTH analogs, such as PTH(3-34), PTH(7-34) and PTH(1-31), have been previously found to exhibit selective activation of only a subset of the usual PTHR second messengers (Rixon, R. H., et al.,
J Bone Miner Res
9:1179-1189 (1994), Whitfield, J. F., and Morley, P.,
Trends Pharmacol Sci
16:382-386 (1995), Fujimori, A., et al.,
Endocrinology
128:3032-3039 (1991), Abou-Samra, A. B., et al.,
Endocrinology
135:2588-2594 (1994); Azarani, A., et al.,
J Biol Chem
271:14931-14936 (1996); Chakravarthy, B. R., et al.,
Biochem Beefiest Res Commun
171:1105-1110 (1990); Fujimori, A., et al.,
Endocrinology
130:29-36 (1992); Jouishomme, H., et al.,
Endocrinology
130:53-60 (1992); Janulis, M., et al.,
Endocrinology
133:713-719 (1993)). The hPTH(1-31) analog was reported to activate AC but not PKC and yet to retain striking anabolic effects in ovariectomized rats (Rixon, R. H., et al.,
J Bone Miner Res
9:1179-1189 (1994), Jouishomme, H., et al.,
J Bone Miner Res
9:943-949 (1994)). Confirmation of such selective signaling via human PTHRs would provide important additional rationale for the development of these signal-specific PTH peptides for clinical use. Thus, there is a need in the art for characterization of responses mediated by PTHR.
SUMMARY OF THE INVENTION
The present inventors isolated and characterized numerous subclones of the well-characterized renal epithelial LLC-PK1 cell line that collectively expressed a broad range of stable transfected human PTHRs. It was found that, as with the rat PTHR, the human receptor activates AC maximally at levels of receptor expression far lower than those needed for PLC activation. Further, the temporal pattern and magnitude of PLC activation in these cells is strongly dependent upon the density of cell-surface human PTHRs across a range of expression above that which elicits maximal AC activation. Surprisingly, it was also found that hPTH(1-31) and hPTH(1-34) activate PLC and cytosolic free calcium transients equivalently via the human PTHR and, moreover, that hPTH(1-31) fully induces other, more delayed biologic responses to PTH in these cells that depend upon cAMP-independent signaling pathways. These findings point to a potential physiologic role of PTHR regulation and differential signaling in fashioning the integrated cellular response in tissues such as bone or kidney, and they indicate that the ligand selectivity of human PTHRs may differ from that of other species of PTHRs.
The present inventors also have developed a
Bringhurst F. Richard
Takasu Hisashi
Sterne Kessler Goldstein & Fox P.L.L.C.
The General Hospital Corporation
Ulm John
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