G protein-dependent activation of a phosphoinositide-specific phospholipase C in UMR-106 osteosarcoma cell membranes

M. Babich, K. L. King, R. A. Nissenson

Research output: Contribution to journalArticle

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Abstract

Recent evidence suggests that guanyl nucleotide binding (G) proteins are involved in receptor-mediated bone resorption and in osteoblastic function, but the nature of the G protein coupled to effectors that are involved in these skeletal effects is unknown. The purposes of this study were to determine (1) whether a G protein mediates activation of phosphoinositide-specific phospholipase C in UMR-106 rat osteosarcoma cells, and (2) whether parathyroid hormone (PTH) and a PTH-like protein (PLP) associated with humoral hypercalcemia of malignancy promote GTP-dependent PIP2 hydrolysis. Addition of GTP (10-4 M) or guanosine 5'-0-(3-thiotriphosphate, GTPγS, 10-5 M) to membranes prepared from UMR-106 cells labeled with [3H]myo-inositol increased both [3H]inositol trisphosphate (IP3) and [3H]inositol bisphosphate (IP2) formation. The increases in [3H]IP2 and [3H]IP3 produced by GTP were 8.6- and 4.3-fold, respectively. GTPγS produced a 17.6- and 11.9-fold increase in [3H]IP2 and [3H[IP3, respectively. The stimulatory effects of GTP and GTPγS were dose dependent (GTP ED50 = 3.9 x 10-6 M; GTPγS ED50 = 2.5 x 10-7 M) and progressive over 10 minutes and required the presence of Mg2+. GTP (10-4 M) and GTPγS (10-5 M) decreased membrane [3H]phosphoinositides concomitantly with increased [3H]IP2 and [3H]IP3. The GDP analog guanosine 5'-O-(2-thiodiphosphate, GDPβS) alone did not alter [3H]IP2 or [3H]IP3 production but at 10-4 M blocks the stimulatory effects of GTP and GTPγS. NaF (3 x 10-2 M) produced a 2.8- and 2.0-fold stimulation of [3H]IP2 and [3H]IP3, respectively. In the presence of 10-4 M GTP, bPTH-(1-34) (1 μg/ml) produced an increase in [3H]IP3 and [3H[IP2 of 23.5 ± 3.0% (p < 0.001) and 14.1 ± 2.5% (p < 0.01) within 2 minutes. hPLP-(1-34)amide (1 μg/ml) produced a 19.8 ± 5.3% (p < 0.05) and 13.2 ± 4.8% (p < 0.05) increase in [3H]IP3 and [3H]IP2. We conclude that UMR-106 membranes possess a G protein-sensitive phosphoinositide-specific phospholipase C. Conceivably, this signal transduction pathway contributes to the skeletal actions of PTH and PLP.

Original languageEnglish (US)
Pages (from-to)549-556
Number of pages8
JournalJournal of Bone and Mineral Research
Volume4
Issue number4
StatePublished - 1989
Externally publishedYes

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Phosphoinositide Phospholipase C
Osteosarcoma
Guanosine Triphosphate
GTP-Binding Proteins
Cell Membrane
Parathyroid Hormone-Related Protein
Inositol
Membranes
Guanosine
Bone Resorption
Phosphatidylinositols
Parathyroid Hormone
Amides
Signal Transduction
Carrier Proteins
Hydrolysis
Nucleotides

ASJC Scopus subject areas

  • Surgery

Cite this

G protein-dependent activation of a phosphoinositide-specific phospholipase C in UMR-106 osteosarcoma cell membranes. / Babich, M.; King, K. L.; Nissenson, R. A.

In: Journal of Bone and Mineral Research, Vol. 4, No. 4, 1989, p. 549-556.

Research output: Contribution to journalArticle

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abstract = "Recent evidence suggests that guanyl nucleotide binding (G) proteins are involved in receptor-mediated bone resorption and in osteoblastic function, but the nature of the G protein coupled to effectors that are involved in these skeletal effects is unknown. The purposes of this study were to determine (1) whether a G protein mediates activation of phosphoinositide-specific phospholipase C in UMR-106 rat osteosarcoma cells, and (2) whether parathyroid hormone (PTH) and a PTH-like protein (PLP) associated with humoral hypercalcemia of malignancy promote GTP-dependent PIP2 hydrolysis. Addition of GTP (10-4 M) or guanosine 5'-0-(3-thiotriphosphate, GTPγS, 10-5 M) to membranes prepared from UMR-106 cells labeled with [3H]myo-inositol increased both [3H]inositol trisphosphate (IP3) and [3H]inositol bisphosphate (IP2) formation. The increases in [3H]IP2 and [3H]IP3 produced by GTP were 8.6- and 4.3-fold, respectively. GTPγS produced a 17.6- and 11.9-fold increase in [3H]IP2 and [3H[IP3, respectively. The stimulatory effects of GTP and GTPγS were dose dependent (GTP ED50 = 3.9 x 10-6 M; GTPγS ED50 = 2.5 x 10-7 M) and progressive over 10 minutes and required the presence of Mg2+. GTP (10-4 M) and GTPγS (10-5 M) decreased membrane [3H]phosphoinositides concomitantly with increased [3H]IP2 and [3H]IP3. The GDP analog guanosine 5'-O-(2-thiodiphosphate, GDPβS) alone did not alter [3H]IP2 or [3H]IP3 production but at 10-4 M blocks the stimulatory effects of GTP and GTPγS. NaF (3 x 10-2 M) produced a 2.8- and 2.0-fold stimulation of [3H]IP2 and [3H]IP3, respectively. In the presence of 10-4 M GTP, bPTH-(1-34) (1 μg/ml) produced an increase in [3H]IP3 and [3H[IP2 of 23.5 ± 3.0{\%} (p < 0.001) and 14.1 ± 2.5{\%} (p < 0.01) within 2 minutes. hPLP-(1-34)amide (1 μg/ml) produced a 19.8 ± 5.3{\%} (p < 0.05) and 13.2 ± 4.8{\%} (p < 0.05) increase in [3H]IP3 and [3H]IP2. We conclude that UMR-106 membranes possess a G protein-sensitive phosphoinositide-specific phospholipase C. Conceivably, this signal transduction pathway contributes to the skeletal actions of PTH and PLP.",
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N2 - Recent evidence suggests that guanyl nucleotide binding (G) proteins are involved in receptor-mediated bone resorption and in osteoblastic function, but the nature of the G protein coupled to effectors that are involved in these skeletal effects is unknown. The purposes of this study were to determine (1) whether a G protein mediates activation of phosphoinositide-specific phospholipase C in UMR-106 rat osteosarcoma cells, and (2) whether parathyroid hormone (PTH) and a PTH-like protein (PLP) associated with humoral hypercalcemia of malignancy promote GTP-dependent PIP2 hydrolysis. Addition of GTP (10-4 M) or guanosine 5'-0-(3-thiotriphosphate, GTPγS, 10-5 M) to membranes prepared from UMR-106 cells labeled with [3H]myo-inositol increased both [3H]inositol trisphosphate (IP3) and [3H]inositol bisphosphate (IP2) formation. The increases in [3H]IP2 and [3H]IP3 produced by GTP were 8.6- and 4.3-fold, respectively. GTPγS produced a 17.6- and 11.9-fold increase in [3H]IP2 and [3H[IP3, respectively. The stimulatory effects of GTP and GTPγS were dose dependent (GTP ED50 = 3.9 x 10-6 M; GTPγS ED50 = 2.5 x 10-7 M) and progressive over 10 minutes and required the presence of Mg2+. GTP (10-4 M) and GTPγS (10-5 M) decreased membrane [3H]phosphoinositides concomitantly with increased [3H]IP2 and [3H]IP3. The GDP analog guanosine 5'-O-(2-thiodiphosphate, GDPβS) alone did not alter [3H]IP2 or [3H]IP3 production but at 10-4 M blocks the stimulatory effects of GTP and GTPγS. NaF (3 x 10-2 M) produced a 2.8- and 2.0-fold stimulation of [3H]IP2 and [3H]IP3, respectively. In the presence of 10-4 M GTP, bPTH-(1-34) (1 μg/ml) produced an increase in [3H]IP3 and [3H[IP2 of 23.5 ± 3.0% (p < 0.001) and 14.1 ± 2.5% (p < 0.01) within 2 minutes. hPLP-(1-34)amide (1 μg/ml) produced a 19.8 ± 5.3% (p < 0.05) and 13.2 ± 4.8% (p < 0.05) increase in [3H]IP3 and [3H]IP2. We conclude that UMR-106 membranes possess a G protein-sensitive phosphoinositide-specific phospholipase C. Conceivably, this signal transduction pathway contributes to the skeletal actions of PTH and PLP.

AB - Recent evidence suggests that guanyl nucleotide binding (G) proteins are involved in receptor-mediated bone resorption and in osteoblastic function, but the nature of the G protein coupled to effectors that are involved in these skeletal effects is unknown. The purposes of this study were to determine (1) whether a G protein mediates activation of phosphoinositide-specific phospholipase C in UMR-106 rat osteosarcoma cells, and (2) whether parathyroid hormone (PTH) and a PTH-like protein (PLP) associated with humoral hypercalcemia of malignancy promote GTP-dependent PIP2 hydrolysis. Addition of GTP (10-4 M) or guanosine 5'-0-(3-thiotriphosphate, GTPγS, 10-5 M) to membranes prepared from UMR-106 cells labeled with [3H]myo-inositol increased both [3H]inositol trisphosphate (IP3) and [3H]inositol bisphosphate (IP2) formation. The increases in [3H]IP2 and [3H]IP3 produced by GTP were 8.6- and 4.3-fold, respectively. GTPγS produced a 17.6- and 11.9-fold increase in [3H]IP2 and [3H[IP3, respectively. The stimulatory effects of GTP and GTPγS were dose dependent (GTP ED50 = 3.9 x 10-6 M; GTPγS ED50 = 2.5 x 10-7 M) and progressive over 10 minutes and required the presence of Mg2+. GTP (10-4 M) and GTPγS (10-5 M) decreased membrane [3H]phosphoinositides concomitantly with increased [3H]IP2 and [3H]IP3. The GDP analog guanosine 5'-O-(2-thiodiphosphate, GDPβS) alone did not alter [3H]IP2 or [3H]IP3 production but at 10-4 M blocks the stimulatory effects of GTP and GTPγS. NaF (3 x 10-2 M) produced a 2.8- and 2.0-fold stimulation of [3H]IP2 and [3H]IP3, respectively. In the presence of 10-4 M GTP, bPTH-(1-34) (1 μg/ml) produced an increase in [3H]IP3 and [3H[IP2 of 23.5 ± 3.0% (p < 0.001) and 14.1 ± 2.5% (p < 0.01) within 2 minutes. hPLP-(1-34)amide (1 μg/ml) produced a 19.8 ± 5.3% (p < 0.05) and 13.2 ± 4.8% (p < 0.05) increase in [3H]IP3 and [3H]IP2. We conclude that UMR-106 membranes possess a G protein-sensitive phosphoinositide-specific phospholipase C. Conceivably, this signal transduction pathway contributes to the skeletal actions of PTH and PLP.

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