Cloning, Functional Expression and Role in Cell Growth Regulation of a Hamster 5-HT2 Receptor Subtype
Ellen Van Obberghen-Schilling, Valerie Vouret-Craviari, Richard J. Haslam*, Jean-Claude Chambard, and Jacques Pouyssegur Centre de Biochimie CNRS Pare Valrose 06034 Nice Cedex, France
INTRODUCTION
We have isolated a hamster fibroblast cDNA clone that encodes a serotoninergic receptor whose deduced amino acid sequence displays 94% identity with the rat brain serotonin (5-HT) type 2 receptor. When expressed in Xenopus oocytes, the hamster receptor efficiently couples to the phosphoinositide second messenger system and leads to intracellular Ca2+ mobilization in response to 5-HT. To determine the pharmacological properties of this receptor, and to evaluate the role of phospholipase C (PLC) activation in growth modulation by 5-HT, we have expressed it in hamster fibroblasts. Transfected cells that express 5-HT receptors were selected using a novel method based on coexpression of the Na + /H + antiporter gene as a selectable marker. After cotransfection of the 5-HT receptor and Na + /H + antiporter cDNAs in fibroblasts lacking antiporter activity (variants of the CCL39 line), 50% of the clones resistant to an acute acid load express functional receptors. The pharmacological profile of the transfected receptor is consistent with it being of the 5HT2 subtype, and the extent of 5-HT-stimulated PLC activation in independent clones correlates with their relative level of cRNA expression. In cells in where addition of 5-HT leads to strong activation of PLC, and inhibition of adenylate cyclase via endogenous 5-HT1b receptors, 5-HT alone has little effect on DNA synthesis stimulation. Thus we conclude that activation of the PLC signalling pathway in these cells is not sufficient to trigger G0/G1 to S phase transition. Strong activation of PLC via 5-HT2 receptors does however contribute to the synergy observed between 5-HT (Gi-coupled pathway) and fibroblast growth factor (tyrosine kinase-activated pathway) on DNA synthesis reinitiation in transfected cells. (Molecular Endocrinology 5: 881-889, 1991)
Serotonin (5-HT) is a multifunctional bioamine that binds to and activates multiple receptor subtypes with diverse physiological consequences in the central nervous system and peripheral tissues. Among the 5-HT receptor subtypes whose primary structures have been deduced by cDNA and genomic cloning, there is considerable sequence conservation as well as homology to the larger family of receptors coupled to intracellular effectors by guanine nucleotide binding proteins (G-proteins) (for review see 1). Studies addressing the role of receptor tyrosine kinases and G-protein-mediated events in cellular proliferation have been carried out for several years in our laboratory using the CCL39 line of hamster lung fibroblasts (2-6). These cells express a number of growth factor receptor tyrosine kinases, including receptors for fibroblast growth factor (FGF), platelet-derived growth factor, and epidermal growth factor, as well as receptors for growth-promoting agents such as a-thrombin and 5-HT which activate G-protein-coupled pathways (for review see (7). In CCL39 cells both a-thrombin and 5-HT: 1) activate a phosphatidylinositol bisphosphatespecific phospholipase C (PLC) and 2) inhibit agoniststimulated adenylate cyclase. Whereas the receptor, or receptors, that mediate a-thrombin action have not been identified, 5-HT has been shown to activate 5-HT type 1 b (coupled negatively to adenylate cyclase) and type 2 (coupled to PLC activation) receptors on CCL39 cells. The importance of one or more Gi-coupled signal transduction pathways, sensitive to pertussis toxin, in the mitogenic action of these two ligands has previously been established in CCL39 cells (2, 6). A pertussis toxin-sensitive Gi protein has also been implicated in the mitogenic stimulation of rat fibroblasts by lysophosphatidate (8). Although activation of the PLC/protein kinase C pathway has generally been assumed to play a crucial role in mitogen-stimulated DNA synthesis initiation, recent studies indicate that activation of inositol lipid breakdown is not sufficient to induce GO to S phase progres-
0888-8809/91/0881 -0889$03.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society
881
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 21 November 2015. at 14:22 For personal use only. No other uses without permission. . All rights reserved.
Vol 5 No. 7
MOL ENDO-1991 882
sion (6, 8, 9). In fact, it has been found that strong and persistent activation of PLC in CCL39 cells transfected with a human M1 muscarinic acetylcholine receptor (9), or with a 5-HT1c receptor (10; Kahan C , K. Seuwen, D. Julius, and J. Pouyssegur, in preparation) does not stimulate cell proliferation. In contrast, Julius et al. (11, 12) have reported that expression of both 5-HT1 c and 5-HT2 receptors in NIH 3T3 cells leads to cellular transformation. In the present study we have attempted to further elucidate the role of the PLC signalling pathway in cell growth modulation by 5-HT via 5-HT2 receptors. To do so, we have cloned the hamster 5-HT2 receptor and expressed it in CCL39 cell variants lacking endogenous 5-HT2 receptors using a newly designed selection system for introducing the cloned gene into cells. Clones displaying a strong PLC activation in response to 5-HT were analyzed for their mitogenic response to the drug alone, or together with tyrosine kinase-activating growth factors.
the primary structure of this receptor predicts a protein with seven transmembrane helices, an extracellular Nterminus, and an intracellular C-terminus. As denoted by filled symbols in Fig. 1, considerable sequence identity in the membrane-spanning domains is shared by all of the serotoninergic receptors whose sequences have been deduced by cDNA or genomic cloning including the 5-HT1a (human and rat) (16,17), 5-HT1c (rat) (18), and 5-HT2 (rat) (11, 14) receptors. Among these subtypes, 5-HT2 and 5-HT1c receptors display the highest sequence homology (identical residues are represented by square symbols in Fig. 1). Sequence identity is highest in the transmembrane domains (77%) and extends into portions of the cytoplasmic loops, particularly in the C-terminal portion of the second and third loops, as well as the N-terminal extremity of the cytoplasmic tail. Since both the 5-HT2 and 5-HT1c receptors are coupled to PLC, conservation of the sequences situated intracellularly may suggest a role for them in G-protein recognition and activation.
RESULTS
Expression in Xenopus Oocytes
Primary Structure of the Chinese Hamster Serotoninergic Receptor
As a first step in analyzing the functional properties of the cloned 5-HT receptor, we chose to express it in Xenopus oocytes. Activation of receptors coupled to the PLC signalling pathway in oocytes leads to intracellular Ca2+ mobilization which can be measured by 45 Ca2+ efflux or Ca2+-dependent C\~ channel activation (19-22). In previous studies we have described weak expression of 5-HT receptors in Xenopus oocytes after injection of CCL39 cell mRNA (the same mRNA used for cDNA library construction) using both methods to detect receptor expression (23).
We have recently reported the nucleotide sequence of a Chinese hamster cDNA clone that displays high homology to the rat brain 5-HT type 2 receptor cDNA sequence (11, 13, 14). Both the rat and hamster sequences contain an open reading frame that encodes a 471 amino acid protein, schematically represented in Fig. 1. Based on analogy to visual rhodopsin and other members of the G-protein-coupled receptor family (15),
>©©©H®®®®®©©©®©©®®©©©© ©®©©®©©® ©©' Fig. 1. Primary Structure and Predicted Transmembrane Model of the Chinese Hamster 5-HT Receptor Amino acid identity among the cloned serotoninergic receptors [including human (17) and rat (16) 5-HT1a; rat 5-HT1c (18); and rat 5-HT2 (11, 14)] is represented by solid symbols. Sequence identity with the 5-HT receptors coupled to PLC is denoted by squares.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 21 November 2015. at 14:22 For personal use only. No other uses without permission. . All rights reserved.
883
Functional Expression of 5-HT2 Receptors
The longest 5-HT receptor cDNA clone that we isolated (-3.5 kilobase pairs) contained approximately 1 kilobase pairs of 5'-untranslated sequences, the protein coding region and a 3'-noncoding region of approximately 1 kilobase (kb) pairs ending with a polyadenylation signal and a poly(A+) tail of 15 residues (Fig. 2a) (13). RNA corresponding to the entire receptor cDNA was transcribed in vitro as described in Materials and Methods and injected in oocytes. After 2 days of expression, addition of 10 HM 5-HT induced a 12-fold increase in 45Ca2+ release from cRNA-injected oocytes (Fig. 2b), whereas no stimulation was observed in H2Oinjected or noninjected oocytes (not shown). Injection of total rat brain RNA, our control for 5-HT receptor expression [predominantly 5-HT type 1c receptor (24)], led to a 9-fold stimulation of 45Ca2+ efflux by 5HT. In voltage clamp experiments the application of 5HT to cRNA-injected oocytes induced a large inward current corresponding to the activation of Ca2+-dependent Cl~ channels (not shown). These results confirmed that the cloned hamster cDNA encoded a functional 5HT receptor coupled to intracellular signalling systems that increase intracellular Ca2+. Expression in cultured cells The pharmacological properties of the cloned receptor and biological consequences of its expression were examined in cultured fibroblasts after transfection of
CHSR (3.5 kb)
A.
ec
_
52 sI 11
I I I 500 bp
B. ~
30
5-HT(10" 5 M)
"
Ellen Van Obberghen-Schilling, Valerie Vouret-Craviari, Richard J. Haslam*, Jean-Claude Chambard, and Jacques Pouyssegur Centre de Biochimie CNRS Pare Valrose 06034 Nice Cedex, France
INTRODUCTION
We have isolated a hamster fibroblast cDNA clone that encodes a serotoninergic receptor whose deduced amino acid sequence displays 94% identity with the rat brain serotonin (5-HT) type 2 receptor. When expressed in Xenopus oocytes, the hamster receptor efficiently couples to the phosphoinositide second messenger system and leads to intracellular Ca2+ mobilization in response to 5-HT. To determine the pharmacological properties of this receptor, and to evaluate the role of phospholipase C (PLC) activation in growth modulation by 5-HT, we have expressed it in hamster fibroblasts. Transfected cells that express 5-HT receptors were selected using a novel method based on coexpression of the Na + /H + antiporter gene as a selectable marker. After cotransfection of the 5-HT receptor and Na + /H + antiporter cDNAs in fibroblasts lacking antiporter activity (variants of the CCL39 line), 50% of the clones resistant to an acute acid load express functional receptors. The pharmacological profile of the transfected receptor is consistent with it being of the 5HT2 subtype, and the extent of 5-HT-stimulated PLC activation in independent clones correlates with their relative level of cRNA expression. In cells in where addition of 5-HT leads to strong activation of PLC, and inhibition of adenylate cyclase via endogenous 5-HT1b receptors, 5-HT alone has little effect on DNA synthesis stimulation. Thus we conclude that activation of the PLC signalling pathway in these cells is not sufficient to trigger G0/G1 to S phase transition. Strong activation of PLC via 5-HT2 receptors does however contribute to the synergy observed between 5-HT (Gi-coupled pathway) and fibroblast growth factor (tyrosine kinase-activated pathway) on DNA synthesis reinitiation in transfected cells. (Molecular Endocrinology 5: 881-889, 1991)
Serotonin (5-HT) is a multifunctional bioamine that binds to and activates multiple receptor subtypes with diverse physiological consequences in the central nervous system and peripheral tissues. Among the 5-HT receptor subtypes whose primary structures have been deduced by cDNA and genomic cloning, there is considerable sequence conservation as well as homology to the larger family of receptors coupled to intracellular effectors by guanine nucleotide binding proteins (G-proteins) (for review see 1). Studies addressing the role of receptor tyrosine kinases and G-protein-mediated events in cellular proliferation have been carried out for several years in our laboratory using the CCL39 line of hamster lung fibroblasts (2-6). These cells express a number of growth factor receptor tyrosine kinases, including receptors for fibroblast growth factor (FGF), platelet-derived growth factor, and epidermal growth factor, as well as receptors for growth-promoting agents such as a-thrombin and 5-HT which activate G-protein-coupled pathways (for review see (7). In CCL39 cells both a-thrombin and 5-HT: 1) activate a phosphatidylinositol bisphosphatespecific phospholipase C (PLC) and 2) inhibit agoniststimulated adenylate cyclase. Whereas the receptor, or receptors, that mediate a-thrombin action have not been identified, 5-HT has been shown to activate 5-HT type 1 b (coupled negatively to adenylate cyclase) and type 2 (coupled to PLC activation) receptors on CCL39 cells. The importance of one or more Gi-coupled signal transduction pathways, sensitive to pertussis toxin, in the mitogenic action of these two ligands has previously been established in CCL39 cells (2, 6). A pertussis toxin-sensitive Gi protein has also been implicated in the mitogenic stimulation of rat fibroblasts by lysophosphatidate (8). Although activation of the PLC/protein kinase C pathway has generally been assumed to play a crucial role in mitogen-stimulated DNA synthesis initiation, recent studies indicate that activation of inositol lipid breakdown is not sufficient to induce GO to S phase progres-
0888-8809/91/0881 -0889$03.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society
881
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 21 November 2015. at 14:22 For personal use only. No other uses without permission. . All rights reserved.
Vol 5 No. 7
MOL ENDO-1991 882
sion (6, 8, 9). In fact, it has been found that strong and persistent activation of PLC in CCL39 cells transfected with a human M1 muscarinic acetylcholine receptor (9), or with a 5-HT1c receptor (10; Kahan C , K. Seuwen, D. Julius, and J. Pouyssegur, in preparation) does not stimulate cell proliferation. In contrast, Julius et al. (11, 12) have reported that expression of both 5-HT1 c and 5-HT2 receptors in NIH 3T3 cells leads to cellular transformation. In the present study we have attempted to further elucidate the role of the PLC signalling pathway in cell growth modulation by 5-HT via 5-HT2 receptors. To do so, we have cloned the hamster 5-HT2 receptor and expressed it in CCL39 cell variants lacking endogenous 5-HT2 receptors using a newly designed selection system for introducing the cloned gene into cells. Clones displaying a strong PLC activation in response to 5-HT were analyzed for their mitogenic response to the drug alone, or together with tyrosine kinase-activating growth factors.
the primary structure of this receptor predicts a protein with seven transmembrane helices, an extracellular Nterminus, and an intracellular C-terminus. As denoted by filled symbols in Fig. 1, considerable sequence identity in the membrane-spanning domains is shared by all of the serotoninergic receptors whose sequences have been deduced by cDNA or genomic cloning including the 5-HT1a (human and rat) (16,17), 5-HT1c (rat) (18), and 5-HT2 (rat) (11, 14) receptors. Among these subtypes, 5-HT2 and 5-HT1c receptors display the highest sequence homology (identical residues are represented by square symbols in Fig. 1). Sequence identity is highest in the transmembrane domains (77%) and extends into portions of the cytoplasmic loops, particularly in the C-terminal portion of the second and third loops, as well as the N-terminal extremity of the cytoplasmic tail. Since both the 5-HT2 and 5-HT1c receptors are coupled to PLC, conservation of the sequences situated intracellularly may suggest a role for them in G-protein recognition and activation.
RESULTS
Expression in Xenopus Oocytes
Primary Structure of the Chinese Hamster Serotoninergic Receptor
As a first step in analyzing the functional properties of the cloned 5-HT receptor, we chose to express it in Xenopus oocytes. Activation of receptors coupled to the PLC signalling pathway in oocytes leads to intracellular Ca2+ mobilization which can be measured by 45 Ca2+ efflux or Ca2+-dependent C\~ channel activation (19-22). In previous studies we have described weak expression of 5-HT receptors in Xenopus oocytes after injection of CCL39 cell mRNA (the same mRNA used for cDNA library construction) using both methods to detect receptor expression (23).
We have recently reported the nucleotide sequence of a Chinese hamster cDNA clone that displays high homology to the rat brain 5-HT type 2 receptor cDNA sequence (11, 13, 14). Both the rat and hamster sequences contain an open reading frame that encodes a 471 amino acid protein, schematically represented in Fig. 1. Based on analogy to visual rhodopsin and other members of the G-protein-coupled receptor family (15),
>©©©H®®®®®©©©®©©®®©©©© ©®©©®©©® ©©' Fig. 1. Primary Structure and Predicted Transmembrane Model of the Chinese Hamster 5-HT Receptor Amino acid identity among the cloned serotoninergic receptors [including human (17) and rat (16) 5-HT1a; rat 5-HT1c (18); and rat 5-HT2 (11, 14)] is represented by solid symbols. Sequence identity with the 5-HT receptors coupled to PLC is denoted by squares.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 21 November 2015. at 14:22 For personal use only. No other uses without permission. . All rights reserved.
883
Functional Expression of 5-HT2 Receptors
The longest 5-HT receptor cDNA clone that we isolated (-3.5 kilobase pairs) contained approximately 1 kilobase pairs of 5'-untranslated sequences, the protein coding region and a 3'-noncoding region of approximately 1 kilobase (kb) pairs ending with a polyadenylation signal and a poly(A+) tail of 15 residues (Fig. 2a) (13). RNA corresponding to the entire receptor cDNA was transcribed in vitro as described in Materials and Methods and injected in oocytes. After 2 days of expression, addition of 10 HM 5-HT induced a 12-fold increase in 45Ca2+ release from cRNA-injected oocytes (Fig. 2b), whereas no stimulation was observed in H2Oinjected or noninjected oocytes (not shown). Injection of total rat brain RNA, our control for 5-HT receptor expression [predominantly 5-HT type 1c receptor (24)], led to a 9-fold stimulation of 45Ca2+ efflux by 5HT. In voltage clamp experiments the application of 5HT to cRNA-injected oocytes induced a large inward current corresponding to the activation of Ca2+-dependent Cl~ channels (not shown). These results confirmed that the cloned hamster cDNA encoded a functional 5HT receptor coupled to intracellular signalling systems that increase intracellular Ca2+. Expression in cultured cells The pharmacological properties of the cloned receptor and biological consequences of its expression were examined in cultured fibroblasts after transfection of
CHSR (3.5 kb)
A.
ec
_
52 sI 11
I I I 500 bp
B. ~
30
5-HT(10" 5 M)
"
