Differentiation-dependent expression of catecholamine-stimulated adenylate cyclase. Roles of the β-receptor and G/F protein in differentiating 3T3-L1 adipocytes

E. Lai, O. M. Rosen, C. S. Rubin

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Abstract

3T3-L1 Preadipocytes possess an adenylate cyclase system that is highly stimulated (~4.5-fold) by 10 μM GTP in the absence of β-adrenergic agonists and other hormones. During the differentiation of preadipocytes into adipocytes, a sharp decline in this activity precedes the manifestation of catecholamine-stimulated (6- to 10-fold) adenylate cyclase activity. Preadipocytes exhibit 1900 high affinity β-receptors/cell, but the maximal occupation of these receptors by isoproterenol results in only a 20% elevation in the rate of cAMP synthesis. During differentiation, β-receptor number increases by only 60-70% to 3100 sites/cell. β-Receptors in preadipocyte membranes have a K(D) of 0.2 nM for [125I]iodohyroxybenzylpindolol and decrease their affinity for β-agonists in the presence of GTP. During prolonged treatment with 5μM isoproterenol, preadipocytes down regulate more than 80% of their receptors. No significant changes in these properties occurred during adipocyte development. Moreover, β-receptors in preadipocyte and adipocyte membranes exhibited similar affinities and stereoselectivities for a series of β-agonists and β-antagonists. In contrast to the unaltered properties of the β-receptor, the regulation of adenylate cyclase activity by GTP changes markedly during adipocyte development. GTP (10 μM) stimulates the preadipocyte enzyme equally well in the absence or presence of isoproterenol, while the GTP-mediated enhancement of adipocyte membrane adenylate cyclase activity is highly dependent on the presence of the β-adrenergic agonist. These observations suggested that G/F protein exhibited different sets of regulatory (coupling) properties in the presence of similar β-receptors in undifferentiated and differentiated cells. In complementary experiments, the cholera toxin-catalyzed [32P]ADP-ribosylation of 3T3-L1 membranes revealed dramatic differences between the G/F subunits of preadipocytes and adipocytes. In adipocyte membranes, 13-fold more 32P was incorporated into the 42,000-dalton component and 4-fold more labeling was observed in the 49,000- to 50,000-dalton doublet than in the corresponding G/F subunits in preadipocyte membranes. Furthermore, determinations of the relative proportions of the two 32P-labeled components of G/F disclosed a preponderance (4.2:1) of the 49,000- to 50,000-dalton species in preadipocyte membranes and nearly equal amounts of the 42,000-dalton and 49,000- to 50,000-dalton polypeptides in adipocyte membranes. These findings raise the possibility that differentiation-associated changes in the regulation and coupling properties of catecholamine-stimulated adenylate cyclase may be determined principally by modulation of the levels, proportions, and/or properties of the constituents of G/F.

Original languageEnglish (US)
Pages (from-to)12866-12874
Number of pages9
JournalJournal of Biological Chemistry
Volume256
Issue number24
StatePublished - 1981

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Adenylyl Cyclases
Adipocytes
Catecholamines
Membranes
Guanosine Triphosphate
Proteins
Isoproterenol
Adrenergic Agonists
Stereoselectivity
Cholera Toxin
Occupations
Adenosine Diphosphate
Labeling
Down-Regulation
Modulation
Hormones
Peptides
Enzymes

ASJC Scopus subject areas

  • Biochemistry

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Differentiation-dependent expression of catecholamine-stimulated adenylate cyclase. Roles of the β-receptor and G/F protein in differentiating 3T3-L1 adipocytes. / Lai, E.; Rosen, O. M.; Rubin, C. S.

In: Journal of Biological Chemistry, Vol. 256, No. 24, 1981, p. 12866-12874.

Research output: Contribution to journalArticle

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abstract = "3T3-L1 Preadipocytes possess an adenylate cyclase system that is highly stimulated (~4.5-fold) by 10 μM GTP in the absence of β-adrenergic agonists and other hormones. During the differentiation of preadipocytes into adipocytes, a sharp decline in this activity precedes the manifestation of catecholamine-stimulated (6- to 10-fold) adenylate cyclase activity. Preadipocytes exhibit 1900 high affinity β-receptors/cell, but the maximal occupation of these receptors by isoproterenol results in only a 20{\%} elevation in the rate of cAMP synthesis. During differentiation, β-receptor number increases by only 60-70{\%} to 3100 sites/cell. β-Receptors in preadipocyte membranes have a K(D) of 0.2 nM for [125I]iodohyroxybenzylpindolol and decrease their affinity for β-agonists in the presence of GTP. During prolonged treatment with 5μM isoproterenol, preadipocytes down regulate more than 80{\%} of their receptors. No significant changes in these properties occurred during adipocyte development. Moreover, β-receptors in preadipocyte and adipocyte membranes exhibited similar affinities and stereoselectivities for a series of β-agonists and β-antagonists. In contrast to the unaltered properties of the β-receptor, the regulation of adenylate cyclase activity by GTP changes markedly during adipocyte development. GTP (10 μM) stimulates the preadipocyte enzyme equally well in the absence or presence of isoproterenol, while the GTP-mediated enhancement of adipocyte membrane adenylate cyclase activity is highly dependent on the presence of the β-adrenergic agonist. These observations suggested that G/F protein exhibited different sets of regulatory (coupling) properties in the presence of similar β-receptors in undifferentiated and differentiated cells. In complementary experiments, the cholera toxin-catalyzed [32P]ADP-ribosylation of 3T3-L1 membranes revealed dramatic differences between the G/F subunits of preadipocytes and adipocytes. In adipocyte membranes, 13-fold more 32P was incorporated into the 42,000-dalton component and 4-fold more labeling was observed in the 49,000- to 50,000-dalton doublet than in the corresponding G/F subunits in preadipocyte membranes. Furthermore, determinations of the relative proportions of the two 32P-labeled components of G/F disclosed a preponderance (4.2:1) of the 49,000- to 50,000-dalton species in preadipocyte membranes and nearly equal amounts of the 42,000-dalton and 49,000- to 50,000-dalton polypeptides in adipocyte membranes. These findings raise the possibility that differentiation-associated changes in the regulation and coupling properties of catecholamine-stimulated adenylate cyclase may be determined principally by modulation of the levels, proportions, and/or properties of the constituents of G/F.",
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N2 - 3T3-L1 Preadipocytes possess an adenylate cyclase system that is highly stimulated (~4.5-fold) by 10 μM GTP in the absence of β-adrenergic agonists and other hormones. During the differentiation of preadipocytes into adipocytes, a sharp decline in this activity precedes the manifestation of catecholamine-stimulated (6- to 10-fold) adenylate cyclase activity. Preadipocytes exhibit 1900 high affinity β-receptors/cell, but the maximal occupation of these receptors by isoproterenol results in only a 20% elevation in the rate of cAMP synthesis. During differentiation, β-receptor number increases by only 60-70% to 3100 sites/cell. β-Receptors in preadipocyte membranes have a K(D) of 0.2 nM for [125I]iodohyroxybenzylpindolol and decrease their affinity for β-agonists in the presence of GTP. During prolonged treatment with 5μM isoproterenol, preadipocytes down regulate more than 80% of their receptors. No significant changes in these properties occurred during adipocyte development. Moreover, β-receptors in preadipocyte and adipocyte membranes exhibited similar affinities and stereoselectivities for a series of β-agonists and β-antagonists. In contrast to the unaltered properties of the β-receptor, the regulation of adenylate cyclase activity by GTP changes markedly during adipocyte development. GTP (10 μM) stimulates the preadipocyte enzyme equally well in the absence or presence of isoproterenol, while the GTP-mediated enhancement of adipocyte membrane adenylate cyclase activity is highly dependent on the presence of the β-adrenergic agonist. These observations suggested that G/F protein exhibited different sets of regulatory (coupling) properties in the presence of similar β-receptors in undifferentiated and differentiated cells. In complementary experiments, the cholera toxin-catalyzed [32P]ADP-ribosylation of 3T3-L1 membranes revealed dramatic differences between the G/F subunits of preadipocytes and adipocytes. In adipocyte membranes, 13-fold more 32P was incorporated into the 42,000-dalton component and 4-fold more labeling was observed in the 49,000- to 50,000-dalton doublet than in the corresponding G/F subunits in preadipocyte membranes. Furthermore, determinations of the relative proportions of the two 32P-labeled components of G/F disclosed a preponderance (4.2:1) of the 49,000- to 50,000-dalton species in preadipocyte membranes and nearly equal amounts of the 42,000-dalton and 49,000- to 50,000-dalton polypeptides in adipocyte membranes. These findings raise the possibility that differentiation-associated changes in the regulation and coupling properties of catecholamine-stimulated adenylate cyclase may be determined principally by modulation of the levels, proportions, and/or properties of the constituents of G/F.

AB - 3T3-L1 Preadipocytes possess an adenylate cyclase system that is highly stimulated (~4.5-fold) by 10 μM GTP in the absence of β-adrenergic agonists and other hormones. During the differentiation of preadipocytes into adipocytes, a sharp decline in this activity precedes the manifestation of catecholamine-stimulated (6- to 10-fold) adenylate cyclase activity. Preadipocytes exhibit 1900 high affinity β-receptors/cell, but the maximal occupation of these receptors by isoproterenol results in only a 20% elevation in the rate of cAMP synthesis. During differentiation, β-receptor number increases by only 60-70% to 3100 sites/cell. β-Receptors in preadipocyte membranes have a K(D) of 0.2 nM for [125I]iodohyroxybenzylpindolol and decrease their affinity for β-agonists in the presence of GTP. During prolonged treatment with 5μM isoproterenol, preadipocytes down regulate more than 80% of their receptors. No significant changes in these properties occurred during adipocyte development. Moreover, β-receptors in preadipocyte and adipocyte membranes exhibited similar affinities and stereoselectivities for a series of β-agonists and β-antagonists. In contrast to the unaltered properties of the β-receptor, the regulation of adenylate cyclase activity by GTP changes markedly during adipocyte development. GTP (10 μM) stimulates the preadipocyte enzyme equally well in the absence or presence of isoproterenol, while the GTP-mediated enhancement of adipocyte membrane adenylate cyclase activity is highly dependent on the presence of the β-adrenergic agonist. These observations suggested that G/F protein exhibited different sets of regulatory (coupling) properties in the presence of similar β-receptors in undifferentiated and differentiated cells. In complementary experiments, the cholera toxin-catalyzed [32P]ADP-ribosylation of 3T3-L1 membranes revealed dramatic differences between the G/F subunits of preadipocytes and adipocytes. In adipocyte membranes, 13-fold more 32P was incorporated into the 42,000-dalton component and 4-fold more labeling was observed in the 49,000- to 50,000-dalton doublet than in the corresponding G/F subunits in preadipocyte membranes. Furthermore, determinations of the relative proportions of the two 32P-labeled components of G/F disclosed a preponderance (4.2:1) of the 49,000- to 50,000-dalton species in preadipocyte membranes and nearly equal amounts of the 42,000-dalton and 49,000- to 50,000-dalton polypeptides in adipocyte membranes. These findings raise the possibility that differentiation-associated changes in the regulation and coupling properties of catecholamine-stimulated adenylate cyclase may be determined principally by modulation of the levels, proportions, and/or properties of the constituents of G/F.

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