Regulation of Adenylyl Cyclase Activity by /?-Adrenergic Agonists in a Desensitization-Resistant Mutant Cell Line
Michael F. Olson*, Jennivine Tsao, Douglas J. Pont, and Bernard P. Schimmer Banting and Best Department of Medical Research Department of Pharmacology University of Toronto Toronto, Ontario, Canada M5G 1L6
INTRODUCTION
Mutant clones resistant to ACTH-induced desensitization of adenylyl cyclase (Y1DR) were previously isolated from the Y1 mouse adrenocortical tumor cell line. In this study, both parental Y1 cells (Y1DS) and a Y1 DR mutant were transfected with a gene encoding the mouse #2-adrenergic receptor, and transfectants isolated from both Y1 DS and Y1DR cells were shown to express /?2-adrenergic receptors. These transfectants responded to the #-adrenergic agonist isoproterenol with increases in adenylyl cyclase activity and steroidogenesis and changes in cell shape. The transfectants were analyzed to determine whether the Y1 DR mutation was specific for ACTH-induced desensitization of adenylyl cyclase or also affected desensitization of adenylyl cyclase via the £2-adrenergic receptor. Treatment of intact Y1 DS transfectants with isoproterenol caused a rapid desensitization of the adenylyl cyclase system to further stimulation by the /?-adrenergic agonist. Treatment of intact cells with isoproterenol did not affect ACTH-stimulated adenylyl cyclase activity, indicating that desensitization was agonist specific or homologous. Y1 DR transfectants were resistant to the desensitizing effects of isoproterenol in intact cells as well as in cell homogenates. These results indicate that the mutation in Y1 DR transfectants affects a component that is common to the pathways of isoproterenol-induced desensitization and ACTHinduced desensitization of adenylyl cyclase. As determined using the hydrophilic 0-receptor antagonist CGP-12177, isoproterenol caused a rapid sequestration of cell surface receptors in both Y1 DS and Y1 DR transfectants. From these results we infer that the DR phenotype does not arise from mutations affecting receptor sequestration and that receptor number does not limit the response to isoproterenol in these transfectants. (Molecular Endocrinology 5: 34-41, 1991)
Many hormones and neurotransmitters regulate cell function by acutely stimulating adenylyl cyclase and increasing intracellular cAMP. Chronic stimulation by these agents, however, often leads to a diminution of their cellular effectiveness and is associated with a dampened effect on the adenylyl cyclase system. This latter phenomenon, termed agonist-induced desensitization, has been extensively investigated in /3-adrenergic responsive systems and is thought to involve at least four distinct events associated with receptor regulation: uncoupling of the hormone receptor from adenylyl cyclase, phosphorylation of the uncoupled receptor, intemalization of the phosphorylated receptor, and receptor degradation. Dephosphorylation of the receptor is thought to be required for the recycling and resensitization of the system (1). During our studies on regulation of adenylyl cyclase in Y1 mouse adrenocortical tumor cells by the peptide hormone ACTH, we identified a class of mutants that responded to ACTH with increased adenylyl cyclase activity, but resisted the desensitizing effects of the hormone (2). These mutants offer potentially valuable tools to explore the mechanisms involved in desensitization of adenylyl cyclase by peptide hormones. By defining the biochemical and molecular bases of these mutations, we should be able to identify obligatory components of the system. In this study we transfected both parental Y1 cells (Y1DS) and a desensitization-resistant mutant (Y1DR) with the gene encoding the mouse /32-adrenergic receptor (/32-AR) and examined the transformants for desensitization induced by j8-adrenergic agonists. These experiments were carried out in part to assess the extent to which models developed for desensitization induced by /3-adrenergic agonists can be generalized to other hormone-responsive systems. We demonstrate that the mutation affecting ACTH-induced desensitization in Y1 DR clones also affects desensitization via the trans-
0888-8809/91/0034-0041 $02.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society
34
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 June 2016. at 10:44 For personal use only. No other uses without permission. . All rights reserved.
Desensitization-Resistant Mutants
35
IPR (2
fected /VAR gene and suggest that the two desensitization pathways converge.
RESULTS Isolation of Transfectants Parental Y1 DS and Y1 DR cells were cotransfected with cloned genomic DNA encoding the mouse /32-AR and pSV2-neo, a bacterial gene that confers resistance to neomycin and its analogs. Stable transfectants were isolated after selective growth of the cells in medium supplemented with the neomycin analog G418. As demonstrated previously (3), approximately 0.1% of the cells were able to take up and express the neomycin resistance gene under the conditions described. Transformation-competent cells then were screened for expression of functional /32-ARs based on their ability to round up in the presence of the /3-adrenergic agonist isoproterenol (IPR). This morphological screening procedure was based on the observation that Y1 cells typically grow as flat adherent cells in monolayer culture and become spherical in shape when treated with agents that raise the intracellular levels of cAMP (4). Untransfected Y1 DS and Y1 DR cells did not change cell shape when treated with IPR (e.g. Fig. 1), whereas approximately 75% of the transformation-competent isolates rounded in response to IPR. I PR-responsive isolates were recovered using cloning cylinders and recloned where necessary to establish clonal /32-AR+ lines. Figure 1 demonstrates the characteristic cell rounding effects of IPR in typical /?2-AR+ transfectants of Y1 DS and Y1 DR . The /32-AR+ transfectants also acquired the ability to respond to IPR with increases in steroidogenesis that approached the levels achieved with ACTH or 8-bromo-cAMP (data not shown). Adenylyl Cyclase Activities of /?2-AR+ Transfectants The transfectants that responded to IPR with changes in cell shape and increases in steroid synthesis also acquired adenylyl cyclases responsive to IPR. As shown in Table 1, adenylyl cyclase activities in homogenates of untransfected Y1 DS and Y1 DR cells were stimulated by ACTH and NaF, but were unresponsive to IPR. In Y1 DR cells, the responses to ACTH and NaF were approximately equivalent and represented a 22fold increase in activity over unstimulated levels. In Y1 DS cells, the response to ACTH was approximately two thirds the response to NaF. These differences in relative responses to ACTH and NaF seem to correlate with the Y1 DR and Y1 DS phenotypes, as noted previously (2, 5). Table 1 also shows the effects of ACTH, NaF, and IPR on representative /32-AR+ isolates from transfected Y1 DS (clone DS1-1) and Y1 DR (clone DR6-1) cells. In these transfectants, adenylyl cyclase activities were stimulated by ACTH and NaF to the same levels as in the untransfected parents. In addition, enzyme activities in the transfectants were increased by IPR. Interest-
Y1
DS1-1
Fig. 1. Effects of IPR on the Morphology of DS and DR Transfectants Parental Y1 DS cells and the /32-ARf transfectants DS1 -1 and DR6-1 were incubated in the absence (-) or presence (+) of 2 HM IPR for 1.5 h and photographed under phase contrast microscopy at the magnification indicated in the upper left panel.
ingly, the response of the adenylyl cyclase to IPR in clone DR6-1 was at least twice the response in clone DS1-1 and approached the responses achieved with ACTH and NaF. To determine the extent of variation in the responsiveness of DS and DR transformants to IPR, adenylyl cyclase activities were measured in 43 independent clonal isolates selected on the basis of their morphological response to IPR. Results were expressed as the ratios of the response to IPR and the response to NaF to minimize variations caused by differences in enzyme preparation or enzyme stability among the clones (2). All of the transfectants responded to IPR with increases in adenylyl cyclase activity above the basal levels (Fig.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 June 2016. at 10:44 For personal use only. No other uses without permission. . All rights reserved.
Vol 5 No. 1
MOL ENDO-1991 36
Table 1. Adenylyl cyclase activity in /32-AR+ transfectants Adenylyl cyclase activity Cell Line Y10S DS1-1 Y1DR
DR6-1
NaF
Basal
IPR
ACTH
40 ± 5 95 ±20 50 ±20 90 ±30
40 ± 10 440 ± 60 35 ± 15 955 ± 55
785 ±130 710 ±100 1110 ±130
1020 1300 1100 1300
1180 ± 110
±190 ±175 ±195 ±95
Adenylyl cyclase activity was measured in cell homogenates as described in Materials and Methods. IPR (100 MM). ACTH (20 and NaF (15 ITIM) were added as indicated. Results are expressed as pmol cAMP accumulated/5 min/mg protein ± S.E.M.
£
1.25 1.00 0.75
Inn
0.50
JUn
0.25
0.00
Q
Q
Q
Q
QQ
DO
Q O
Clones Fig. 2.1 PR-Stimulated Adenylyl Cyclase Activity in /32-AR+-Transfected DS and DR Clones Homogenates were prepared from independent /32-AR+ transfectants of Y1 DS and Y1 DR and assayed for adenylyl cyclase activity in the presence of 20 »M IPR or 15 mM NaF, as described in Materials and Methods. /82-AR+ transfectants of Y1 DS have the prefix DS; /32-AR+ transfectants of Y1 DR have the prefix DR. Adenylyl cyclase activity is expressed as a ratio of the response to IPR and NaF in order to correct for differences in sample preparation and enzyme stability among clones. The relative activities achieved with untransfected Y1 DS and Y1 DR cells are equivalent to unstimulated levels of enzyme activity.
2). The basal levels of activity are indicated by the ratios obtained for untransfected Y1 DS and Y1 DR cells (Fig. 2). In almost all cases, the DR transfectants were more responsive to IPR than the DS transfectants (Fig. 2). In 13 of the 16 DR transfectants, the responses to IPR were more than half the responses obtained with NaF; in 24 of the 27 DS transfectants, the responses to IPR were less than half the responses obtained with NaF. Receptor Densities in £2-AR+ Clones The densities of /32-ARs were measured in various DS and DR transfectants in order to determine the extent to which the clonal differences in IPR-responsive adenylyl cyclase activities reflected differences in receptor number. Receptor densities were determined in whole cell competitive binding assays, as described in Materials and Methods, and compared with the levels of IPR-responsive adenylyl cyclase activity observed with each isolate. In untransfected Y1 DS and Y1 DR cells, the levels of ftrAR were undetectable (data not shown), whereas in the DR and DS transformants, receptor densities overlapped and ranged from 4-195 fmol/mg protein (Fig. 3). These values corresponded to approx-
imately 2.4 x 103 to 120 x 103 receptors/cell. A DS transfectant with an IPR/NaF activity ratio less than 0.5 exhibited the highest receptor density among the transfectants analyzed, whereas two DR transfectants with IPR/NaF activity ratios less than 0.5 had receptor densities less than 50 fmol/mg protein. As determined by linear regression analysis, a significant correlation (P < 0.005) was observed between receptor density and IPR-responsive adenylyl cyclase activity among 15 different DS and DR transfectants; however, the correlation coefficient was only 0.5, suggesting that receptor density was not the sole determinant of the response to IPR. Agonist-Induced Desensitization of Adenylyl Cyclase Activity in #2-AR+ Transfectants To determine whether the mutation that rendered Y1 DR cells resistant to ACTH-induced desensitization also affected desensitization through the /32-AR, we compared the ability of IPR to desensitize a representative /32-AR+ transfectant from each parent. Intact cells were treated with ACTH-(1-24) (7.5 nM) or IPR (2 M M) for varying periods of time, washed free of agonist, ho-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 June 2016. at 10:44 For personal use only. No other uses without permission. . All rights reserved.
Desensitization-Resistant Mutants
37
p
Michael F. Olson*, Jennivine Tsao, Douglas J. Pont, and Bernard P. Schimmer Banting and Best Department of Medical Research Department of Pharmacology University of Toronto Toronto, Ontario, Canada M5G 1L6
INTRODUCTION
Mutant clones resistant to ACTH-induced desensitization of adenylyl cyclase (Y1DR) were previously isolated from the Y1 mouse adrenocortical tumor cell line. In this study, both parental Y1 cells (Y1DS) and a Y1 DR mutant were transfected with a gene encoding the mouse #2-adrenergic receptor, and transfectants isolated from both Y1 DS and Y1DR cells were shown to express /?2-adrenergic receptors. These transfectants responded to the #-adrenergic agonist isoproterenol with increases in adenylyl cyclase activity and steroidogenesis and changes in cell shape. The transfectants were analyzed to determine whether the Y1 DR mutation was specific for ACTH-induced desensitization of adenylyl cyclase or also affected desensitization of adenylyl cyclase via the £2-adrenergic receptor. Treatment of intact Y1 DS transfectants with isoproterenol caused a rapid desensitization of the adenylyl cyclase system to further stimulation by the /?-adrenergic agonist. Treatment of intact cells with isoproterenol did not affect ACTH-stimulated adenylyl cyclase activity, indicating that desensitization was agonist specific or homologous. Y1 DR transfectants were resistant to the desensitizing effects of isoproterenol in intact cells as well as in cell homogenates. These results indicate that the mutation in Y1 DR transfectants affects a component that is common to the pathways of isoproterenol-induced desensitization and ACTHinduced desensitization of adenylyl cyclase. As determined using the hydrophilic 0-receptor antagonist CGP-12177, isoproterenol caused a rapid sequestration of cell surface receptors in both Y1 DS and Y1 DR transfectants. From these results we infer that the DR phenotype does not arise from mutations affecting receptor sequestration and that receptor number does not limit the response to isoproterenol in these transfectants. (Molecular Endocrinology 5: 34-41, 1991)
Many hormones and neurotransmitters regulate cell function by acutely stimulating adenylyl cyclase and increasing intracellular cAMP. Chronic stimulation by these agents, however, often leads to a diminution of their cellular effectiveness and is associated with a dampened effect on the adenylyl cyclase system. This latter phenomenon, termed agonist-induced desensitization, has been extensively investigated in /3-adrenergic responsive systems and is thought to involve at least four distinct events associated with receptor regulation: uncoupling of the hormone receptor from adenylyl cyclase, phosphorylation of the uncoupled receptor, intemalization of the phosphorylated receptor, and receptor degradation. Dephosphorylation of the receptor is thought to be required for the recycling and resensitization of the system (1). During our studies on regulation of adenylyl cyclase in Y1 mouse adrenocortical tumor cells by the peptide hormone ACTH, we identified a class of mutants that responded to ACTH with increased adenylyl cyclase activity, but resisted the desensitizing effects of the hormone (2). These mutants offer potentially valuable tools to explore the mechanisms involved in desensitization of adenylyl cyclase by peptide hormones. By defining the biochemical and molecular bases of these mutations, we should be able to identify obligatory components of the system. In this study we transfected both parental Y1 cells (Y1DS) and a desensitization-resistant mutant (Y1DR) with the gene encoding the mouse /32-adrenergic receptor (/32-AR) and examined the transformants for desensitization induced by j8-adrenergic agonists. These experiments were carried out in part to assess the extent to which models developed for desensitization induced by /3-adrenergic agonists can be generalized to other hormone-responsive systems. We demonstrate that the mutation affecting ACTH-induced desensitization in Y1 DR clones also affects desensitization via the trans-
0888-8809/91/0034-0041 $02.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society
34
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 June 2016. at 10:44 For personal use only. No other uses without permission. . All rights reserved.
Desensitization-Resistant Mutants
35
IPR (2
fected /VAR gene and suggest that the two desensitization pathways converge.
RESULTS Isolation of Transfectants Parental Y1 DS and Y1 DR cells were cotransfected with cloned genomic DNA encoding the mouse /32-AR and pSV2-neo, a bacterial gene that confers resistance to neomycin and its analogs. Stable transfectants were isolated after selective growth of the cells in medium supplemented with the neomycin analog G418. As demonstrated previously (3), approximately 0.1% of the cells were able to take up and express the neomycin resistance gene under the conditions described. Transformation-competent cells then were screened for expression of functional /32-ARs based on their ability to round up in the presence of the /3-adrenergic agonist isoproterenol (IPR). This morphological screening procedure was based on the observation that Y1 cells typically grow as flat adherent cells in monolayer culture and become spherical in shape when treated with agents that raise the intracellular levels of cAMP (4). Untransfected Y1 DS and Y1 DR cells did not change cell shape when treated with IPR (e.g. Fig. 1), whereas approximately 75% of the transformation-competent isolates rounded in response to IPR. I PR-responsive isolates were recovered using cloning cylinders and recloned where necessary to establish clonal /32-AR+ lines. Figure 1 demonstrates the characteristic cell rounding effects of IPR in typical /?2-AR+ transfectants of Y1 DS and Y1 DR . The /32-AR+ transfectants also acquired the ability to respond to IPR with increases in steroidogenesis that approached the levels achieved with ACTH or 8-bromo-cAMP (data not shown). Adenylyl Cyclase Activities of /?2-AR+ Transfectants The transfectants that responded to IPR with changes in cell shape and increases in steroid synthesis also acquired adenylyl cyclases responsive to IPR. As shown in Table 1, adenylyl cyclase activities in homogenates of untransfected Y1 DS and Y1 DR cells were stimulated by ACTH and NaF, but were unresponsive to IPR. In Y1 DR cells, the responses to ACTH and NaF were approximately equivalent and represented a 22fold increase in activity over unstimulated levels. In Y1 DS cells, the response to ACTH was approximately two thirds the response to NaF. These differences in relative responses to ACTH and NaF seem to correlate with the Y1 DR and Y1 DS phenotypes, as noted previously (2, 5). Table 1 also shows the effects of ACTH, NaF, and IPR on representative /32-AR+ isolates from transfected Y1 DS (clone DS1-1) and Y1 DR (clone DR6-1) cells. In these transfectants, adenylyl cyclase activities were stimulated by ACTH and NaF to the same levels as in the untransfected parents. In addition, enzyme activities in the transfectants were increased by IPR. Interest-
Y1
DS1-1
Fig. 1. Effects of IPR on the Morphology of DS and DR Transfectants Parental Y1 DS cells and the /32-ARf transfectants DS1 -1 and DR6-1 were incubated in the absence (-) or presence (+) of 2 HM IPR for 1.5 h and photographed under phase contrast microscopy at the magnification indicated in the upper left panel.
ingly, the response of the adenylyl cyclase to IPR in clone DR6-1 was at least twice the response in clone DS1-1 and approached the responses achieved with ACTH and NaF. To determine the extent of variation in the responsiveness of DS and DR transformants to IPR, adenylyl cyclase activities were measured in 43 independent clonal isolates selected on the basis of their morphological response to IPR. Results were expressed as the ratios of the response to IPR and the response to NaF to minimize variations caused by differences in enzyme preparation or enzyme stability among the clones (2). All of the transfectants responded to IPR with increases in adenylyl cyclase activity above the basal levels (Fig.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 June 2016. at 10:44 For personal use only. No other uses without permission. . All rights reserved.
Vol 5 No. 1
MOL ENDO-1991 36
Table 1. Adenylyl cyclase activity in /32-AR+ transfectants Adenylyl cyclase activity Cell Line Y10S DS1-1 Y1DR
DR6-1
NaF
Basal
IPR
ACTH
40 ± 5 95 ±20 50 ±20 90 ±30
40 ± 10 440 ± 60 35 ± 15 955 ± 55
785 ±130 710 ±100 1110 ±130
1020 1300 1100 1300
1180 ± 110
±190 ±175 ±195 ±95
Adenylyl cyclase activity was measured in cell homogenates as described in Materials and Methods. IPR (100 MM). ACTH (20 and NaF (15 ITIM) were added as indicated. Results are expressed as pmol cAMP accumulated/5 min/mg protein ± S.E.M.
£
1.25 1.00 0.75
Inn
0.50
JUn
0.25
0.00
Q
Q
Q
Q
DO
Q O
Clones Fig. 2.1 PR-Stimulated Adenylyl Cyclase Activity in /32-AR+-Transfected DS and DR Clones Homogenates were prepared from independent /32-AR+ transfectants of Y1 DS and Y1 DR and assayed for adenylyl cyclase activity in the presence of 20 »M IPR or 15 mM NaF, as described in Materials and Methods. /82-AR+ transfectants of Y1 DS have the prefix DS; /32-AR+ transfectants of Y1 DR have the prefix DR. Adenylyl cyclase activity is expressed as a ratio of the response to IPR and NaF in order to correct for differences in sample preparation and enzyme stability among clones. The relative activities achieved with untransfected Y1 DS and Y1 DR cells are equivalent to unstimulated levels of enzyme activity.
2). The basal levels of activity are indicated by the ratios obtained for untransfected Y1 DS and Y1 DR cells (Fig. 2). In almost all cases, the DR transfectants were more responsive to IPR than the DS transfectants (Fig. 2). In 13 of the 16 DR transfectants, the responses to IPR were more than half the responses obtained with NaF; in 24 of the 27 DS transfectants, the responses to IPR were less than half the responses obtained with NaF. Receptor Densities in £2-AR+ Clones The densities of /32-ARs were measured in various DS and DR transfectants in order to determine the extent to which the clonal differences in IPR-responsive adenylyl cyclase activities reflected differences in receptor number. Receptor densities were determined in whole cell competitive binding assays, as described in Materials and Methods, and compared with the levels of IPR-responsive adenylyl cyclase activity observed with each isolate. In untransfected Y1 DS and Y1 DR cells, the levels of ftrAR were undetectable (data not shown), whereas in the DR and DS transformants, receptor densities overlapped and ranged from 4-195 fmol/mg protein (Fig. 3). These values corresponded to approx-
imately 2.4 x 103 to 120 x 103 receptors/cell. A DS transfectant with an IPR/NaF activity ratio less than 0.5 exhibited the highest receptor density among the transfectants analyzed, whereas two DR transfectants with IPR/NaF activity ratios less than 0.5 had receptor densities less than 50 fmol/mg protein. As determined by linear regression analysis, a significant correlation (P < 0.005) was observed between receptor density and IPR-responsive adenylyl cyclase activity among 15 different DS and DR transfectants; however, the correlation coefficient was only 0.5, suggesting that receptor density was not the sole determinant of the response to IPR. Agonist-Induced Desensitization of Adenylyl Cyclase Activity in #2-AR+ Transfectants To determine whether the mutation that rendered Y1 DR cells resistant to ACTH-induced desensitization also affected desensitization through the /32-AR, we compared the ability of IPR to desensitize a representative /32-AR+ transfectant from each parent. Intact cells were treated with ACTH-(1-24) (7.5 nM) or IPR (2 M M) for varying periods of time, washed free of agonist, ho-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 June 2016. at 10:44 For personal use only. No other uses without permission. . All rights reserved.
Desensitization-Resistant Mutants
37
p
