©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Dopamine-responsive Domain in the N-terminal Sequence of Pit-1
TRANSCRIPTIONAL INHIBITION IN ENDOCRINE CELL TYPES (*)

(Received for publication, December 27, 1994; and in revised form, February 1, 1995)

April M. Lew (§) Harry P. Elsholtz (¶)

From the Department of Clinical Biochemistry and the Banting and Best Diabetes Centre, University of Toronto, Toronto, Ontario M5G 1L5, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The POU transcription factor Pit-1 activates the prolactin gene in pituitary lactotrophs and may integrate responses of the gene to external signals. To study the role of Pit-1 in dopaminergic inhibition of the prolactin gene, we transiently transfected Pit-1 and dopamine D2 receptor vectors into a series of heterologous cell lines and examined dopamine regulation of the prolactin gene promoter. Regulation was Pit-1-dependent in all cell lines tested. Moreover, dopamine responsiveness was cell type-specific: stimulatory in fibroblasts (COS-7) and muscle-type cells (P19/Me(2)SO-induced) and inhibitory in pancreatic endocrine (RIN, InR1-G9) and neural-like (P19/retinoic acid-induced) cells. Because dopaminergic responses in Pit-1-transfected RIN cells paralleled those in pituitary GH4 cells, the islet cell line was used to test for sequences in Pit-1 that mediate negative hormone signals. Dopamine responsiveness of the Pit-1 transactivation domain (residues 8-80) was examined using a chimeric LexA construct. LxPit-1, LxSp1, and Lx-glucocorticoid receptor fusions all activated basal transcription, but only LxPit-1 was regulated by dopamine. Regulatory responses of LxPit-1 and full-length Pit-1 were quantitatively similar. In addition, gain-of-function G mutants that inhibit Pit-1-dependent promoters in GH4 cells also suppressed selectively Pit-1- or LxPit-1-dependent promoters in RIN cells. This demonstrates that Pit-1 can function as a specific target for distinct inhibitory G protein signals. Interestingly, Pit-1 sequences N-terminal to the DNA-binding POU domain appear to be sufficient in mediating regulation by these pathways.


INTRODUCTION

Pit-1 (GHF-1) is a member of the POU class of homeodomain-containing transcription factors and has a critical role in the developmental appearance of specific pituitary cell types (reviewed in (1) and (2) ). The predominant Pit-1 isoform in mammals is 291 amino acids long and consists of a 142-residue C-terminal POU domain that mediates DNA binding as well as homo- and heterodimerization, and an N-terminal sequence important for transactivation(3, 4, 5) . Pit-1 activates transcription of the prolactin (PRL) (^1)gene by binding to specific sites in PRL 5`-sequences(6, 7, 8) . PRL promoter activity in pituitary cells is disrupted by mutation of these sites(9) . Moreover, in vitro analyses of Pit-1 mutants from Snell mice (10) and from hypoprolactinemic patients with combined pituitary hormone deficiency (11, 12) have confirmed the importance of Pit-1 integrity for activation of the PRL gene. Because of its pivotal role in PRL gene transcription, regulation of Pit-1 function in pituitary lactotroph cells could provide an effective mechanism for positive or negative control of PRL production.

The tuberoinfundibular neurons of the hypothalamus project to the pituitary stalk and regulate the release of PRL(13) . Regulation is predominantly inhibitory and is mediated by dopamine (DA) D2 receptors present on lactotroph cells. Activation of D2 receptors leads also to rapid decreases in the incorporation of [^3H]Leu into PRL protein (14) and suppression of PRL gene transcription(15) . We have shown that inhibition of the PRL gene by DA is conferred by proximal promoter sequences and that binding sites for Pit-1 are important in this response(16) . The ability of tandemly linked Pit-1 sites to confer dopaminergic responses in the absence of other PRL promoter elements suggests possible direct effects of DA on Pit-1 function.

To establish the importance of Pit-1 in D2 receptor regulation of the PRL promoter, we have transfected promoter/luciferase constructs into cell lines lacking endogenous Pit-1 and examined DA responsiveness following introduction of recombinant Pit-1. We noted previously (17) that responses of the PRL promoter to D2 receptor activation were not only Pit-1-dependent, but also cell type-specific. Whereas in pituitary GH4 cells high affinity Pit-1-binding sites conferred inhibitory regulation by D2 receptors, in Ltk fibroblasts transfected with Pit-1, regulation at these sites was stimulatory. In this study, we identify cell types in which Pit-1 reconstitutes the negative response of the PRL promoter to activated D2 receptors. Furthermore, by use of chimeric transcription factors, we show that N-terminal sequences of Pit-1 can mimic the negative response of a full-length Pit-1 to DA.


EXPERIMENTAL PROCEDURES

Cell Culture

COS-7 cells (a gift from Dr. A. Keating, University of Toronto) were grown in Ham's F-10 medium containing 12.5% horse serum and 2.5% fetal calf serum. P19 cells (a gift from Dr. V. Giguère, University of Toronto) and pancreatic islet lines RIN 1056A and InR1-G9 (gifts from Dr. D. Drucker, University of Toronto) were cultured in Dulbecco's modified Eagle's medium plus 10% fetal calf serum. Differentiation of P19 cells with Me(2)SO and retinoic acid was performed as described(18) , using 0.75% Me(2)SO and 0.3 µM retinoic acid.

Plasmids

The luciferase reporter plasmid -422P/luciferase contains a 456-base pair fragment of the PRL promoter (positions -422 to +34, relative to the transcription start site) and has been described previously(16) . The vector pRc/RSV (Invitrogen) was used for transient expression of the rat DA D2 receptor (415-amino acid variant) (16) . The 2xLexA/luciferase reporter construct and the expression vector pCMV865 for LxPit-1 have been described(4) ; the chimeric Lex protein contains 72 amino acids of rat Pit-1 (residues 8-80) fused C-terminal to an 87-amino acid LexA DNA-binding domain. LxSp1 contains transactivation sequences from human Sp1 inserted into CMV865. The LxRel plasmid contains a LexA chicken c-Rel fusion subcloned 3` to a spleen necrosis virus long terminal repeat(19) . The LxGR plasmid (20) contains the RSV promoter and expresses a LexA fusion protein containing both the N-terminal transactivation and C-terminal ligand-binding sequences of the human glucocorticoid receptor. pcDNA I expression vectors (Invitrogen) for G and G mutants are described in (21) and (22) , respectively.

Transient Transfections and Luciferase Assays

COS-7, P19, and InR1-G9 cells were transfected using the CaPO(4) method as described(4) . RIN 1056A cells were transfected by electroporation at 300 V and 500 microfarads. Transfections included 10 µg of reporter plasmid, 5 µg of D2 receptor expression vector, and 5 µg of either full-length Pit-1 or LxPit-1 expression vectors or control (empty) vectors. Twenty-four hours after transfection, cells were treated with DA (1 µM final concentration) for 5 h as described(16, 17) . Luciferase activity was measured using a Berthold LB9501 luminometer.


RESULTS AND DISCUSSION

Cell Type-specific Regulation of the PRL Promoter by DA D2 Receptors in Pit-1-transfected Cell Lines

We transiently expressed Pit-1 in a number of heterologous cell lines in an effort to reconstitute the negative DA response characteristic of PRL-producing GH4 cells. The results of transfection experiments from COS-7 (monkey kidney fibroblast), P19 (mouse embryonal carcinoma), RIN (rat insulinoma), and InR1-G9 (hamster glucagonoma) cells are shown in Fig. 1. These cell lines were chosen in part because constitutive activity of transfected PRL promoter constructs was sufficiently high (relative light units were at least 10 times above background) to permit measurement of DA responses in the absence of Pit-1 (described in (17) ). Furthermore, in each of these cell lines, the response to DA was PRL promoter-specific as determined by negative control constructs (e.g. RSV promoter, 3xSp1/RSV minimal promoter) (data not shown).


Figure 1: Cell type-specific and Pit-1-dependent regulation of the PRL promoter by DA in heterologous cell lines. A schematic diagram of the constructs used for transfection is shown. Cell lines were transiently transfected with the PRL promoter construct -422P and an expression vector for the D2 receptor. Each transfection included either the Pit-1 expression vectors or an identical vector lacking the Pit-1 cDNA. Values represent the means ± S.E. for three or more independent experiments. For P19 cells that were differentiated using Me(2)SO or retinoic acid, each experiment indicates a separate differentiation event. Bars illustrate the response to DA; basal promoter activity (with or without Pit-1) has been assigned a value of 1. Activation of basal promoter activity by the addition of Pit-1 was as follows: COS-7 = 13.5 ± 1.4-fold, undifferentiated P19 = 7.1 ± 1.0-fold, Me(2)SO (DMSO)-treated P19 = 5.9 ± 0.8-fold, retinoic acid (RA)-treated P19 = 6.3 ± 1.5-fold, RIN = 2.7 ± 0.4-fold, and InR1-G9 = 4.6 ± 1.2-fold. Significant differences in DA regulation between paired values (with or without Pit-1) are indicated by asterisks (Student's t test):***, p < 0.0001;**, p < 0.004; *, p < 0.007. On the yaxis, 2 indicates a 100% increase, and -2 indicates 50% inhibition. CMV, cytomegalovirus.



In the absence of Pit-1, DA failed to regulate the activity of the -422P PRL promoter in each of the transfected cell lines (Fig. 1). Cotransfection of a Pit-1 vector elevated basal promoter activity (see legend to Fig. 1) and reconstituted DA responsiveness in a cell type-specific manner. In COS-7 cells, PRL promoter function was stimulated about 2-fold by DA, a response similar to that observed in Ltk cells(17) .

Undifferentiated P19 cells provided an interesting system in which D2 receptor activation failed to regulate the PRL promoter in either the absence or presence of Pit-1 (Fig. 1). These cells are induced by Me(2)SO or retinoic acid to differentiate into muscle- and neural-like phenotypes, respectively(18) . Following conversion of P19 cells into muscle-type cells, D2 receptor activation stimulated PRL promoter activity 40-50% in the presence of Pit-1. Because high affinity [^3H]spiperone-binding sites were detectable in undifferentiated P19 cells transiently transfected with D2 receptor expression vector, (^2)these functional differences cannot be explained solely on the basis of D2 receptor expression, processing, or transport to cell membranes. The data could suggest that a signaling step distal to the D2 receptor and dependent on Me(2)SO-induced differentiation is required for the stimulatory transcriptional response to DA. In contrast to these cells, P19 cells exposed to retinoic acid exhibited a consistent inhibitory response to DA that was dependent on Pit-1 (Fig. 1). Together with our earlier data(17) , these results demonstrate that unique patterns of differentiation can lead to opposite transcriptional responses of a specific target gene to D2 receptor activation.

To determine whether Pit-1 would mediate an inhibitory response to DA in cells functionally similar to GH4 cells, we tested two endocrine cell lines of pancreatic origin: RIN and InR1-G9. In the absence of Pit-1, the PRL promoter was weakly active in both pancreatic cell lines, but unresponsive to DA (Fig. 1). Cotransfection of the Pit-1 vector elevated basal PRL promoter activity, but in marked contrast to fibroblasts, mediated a 25% (InR1-G9) to 40% (RIN 1056A) inhibitory response to DA. Expression of Pit-1 in these endocrine cells therefore reconstitutes a pituitary-like signaling pathway that specifically mediates dopaminergic suppression of PRL promoter activity.

The basis for cell type-specific differences in regulation of the PRL promoter by D2 receptors is not yet clear. As activation of transfected D2 receptors leads to a reduction in cAMP in most cell lines studied (16, 23, 24, 25, 26, 27, 28) , regulation of cAMP may not adequately explain the cell type-specific differences in D2 receptor-regulated transcription. By contrast, DA-induced changes in [Ca](i) and/or phospholipid turnover do parallel more closely the cell-specific pattern of regulation observed for the PRL promoter. In fibroblasts, D2 receptor activation stimulates accumulation of inositol phosphates (24) , elevates [Ca](i)(17, 24) , and activates PRL promoter activity(17) . It is noteworthy that these responses are characteristic also of GH4 cells treated with stimulatory hormones such as TRH(29) . Furthermore, multimerized Pit-1-binding sites confer stimulation in both DA-treated fibroblasts and TRH-treated GH4 cells (17, 30, 31) , suggesting a similar regulatory mechanism at the transcriptional level. In contrast to fibroblasts, pituitary lactotrophs and GH4ZR7 cells treated with DA exhibit reduced [Ca](i) due to inhibition of voltage-dependent L-type Ca channels(17, 24, 32) . L-type Ca channels are present in other excitable cell types, including pancreatic endocrine cells, and may contribute to the differential pattern of DA-regulated transcription in fibroblast and islet cell lines. Interestingly, in P19 cells, both Me(2)SO and retinoic acid induce excitable cell phenotypes(18) ; however, only in the neurally differentiated cells was an inhibitory Pit-1-dependent response observed after DA treatment.

Identification of a DA-responsive Domain in Pit-1

In GH4 cells, optimal inhibition of the PRL promoter by DA is observed in the native promoter context(16) , suggesting participation of multiple regulatory elements in this response. However, in heterologous endocrine cells, dopaminergic suppression of the PRL promoter is entirely dependent on the presence of Pit-1 (Fig. 1). Moreover, at least 50% of the maximum inhibitory response to DA in GH4 cells can be achieved using three copies of a Pit-1 site linked to a minimal promoter(17) . These data indicate a central role for Pit-1 in integrating the DA response and also show that Pit-1 sites can mediate transcriptional inhibition by DA independently of other promoter elements. We therefore sought to determine how Pit-1 function might be altered by activation of DA D2 receptors.

Extracellular signals, including DA, could affect Pit-1-dependent transcription by one or more mechanisms. First, Pit-1 levels could be altered by regulation of the pit-1 gene promoter (33, 34, 35) or of Pit-1 protein stability(36) . In vivo, this mechanism may be most important in a developmental context, contributing to the establishment of specific anterior pituitary cell types. Second, Pit-1 interactions at coregulatory DNA sites could be affected. This is best illustrated by the hormone-dependent synergism between Pit-1 and members of the nuclear hormone receptor family, including estrogen (37, 38) and retinoic acid (34) receptors. Pit-1 interactions with factors of other classes can also coordinate stimulatory hormone signals(39, 40) . Third, Pit-1 can be phosphorylated by specific protein kinases, resulting in selective changes in DNA binding activity. The predominant acceptor sites are Ser-115 and Thr-220, with the latter (near the N terminus of the homeodomain) being critical for phosphorylation-dependent DNA interaction(41) . In the case of the thyrotropin beta-subunit gene, phosphorylation-enhanced binding of Pit-1 to the thyrotropin beta promoter provides an interesting model for TRH-stimulated transcription(42) .

Of these three mechanisms, we cannot exclude the involvement of a coregulatory interaction in the DA response as co-occupancy can occur on Pit-1-binding sites as short as 26 base pairs (e.g. see (5) ). However, as determined by mobility shift assays and as discussed previously(17) , DA does not detectably alter Pit-1 levels or binding to high affinity sites within the 5-h time period required for maximal transcriptional inhibition. This could suggest that DA suppresses the transactivating function of Pit-1, rather than regulating its DNA binding activity. As the major transactivation domain of Pit-1 has been localized to the N-terminal region(3, 4) , a chimeric transcription factor containing Pit-1 residues 8-80 fused to the Escherichia coli LexA DNA-binding domain (LxPit-1) was tested for DA responsiveness. Regulation was monitored using a reporter plasmid that contained two LexA operator sequences. To ensure that regulation was specific for Pit-1 N-terminal sequences, and not the LexA domain, a number of chimeric LexA transcription factors (Fig. 2A) were compared to LxPit-1. In addition, chimeras were tested in multiple cell lines to determine whether Pit-1 N-terminal sequences might contribute to the characteristic cell-specific pattern of regulation observed with full-length Pit-1.


Figure 2: Dopaminergic regulation of LexA operator-dependent transactivation. A, transcription units of the luciferase reporter and chimeric LexA expression vectors are illustrated. Plasmids are described under ``Experimental Procedures'' and the references cited. Lex-DBD represents the LexA DNA-binding domain. Each cell line was transiently transfected with 10 µg of 2xLexA/luciferase, 5 µg of D2 receptor expression vector, and 5 µg of either the LexA fusion construct or an empty vector(-) as indicated in B-D. CMV, cytomegalovirus; LTR, long terminal repeat; GR, glucocorticoid receptor; RSV, Rous sarcoma virus. B, responsiveness of LexA fusion constructs to DA in COS-7 cells. Cells transfected with the LxGR fusion expression vectors were first treated with 0.1 µM dexamethasone (Dex) or vehicle for 10 min, followed by DA (1 µM final concentration) for 5 h. Each value represents the means ± S.E. of three or more independent experiments. ***, p < 0.0001 versus empty vector control (Student's t test). Activation of constitutive 2xLexA/luciferase activity by the LexA fusion constructs was as follows: LxPit-1 = 6.1 ± 0.9-fold, LxGR = 1.6 ± 0.4-fold, LxGR + dexamethasone = 7.3 ± 1.0-fold, LxRel = 18.6 ± 3.1-fold, and LxSp1 = 13.1 ± 1.1-fold. C, responsiveness of LexA fusion constructs to DA in undifferentiated (leftpanel) and Me(2)SO-treated (right panel) P19 cells. Values represents the means ± S.E. of three or more independent experiments. Left panel, activation of constitutive 2xLexA/luciferase activity in undifferentiated P19 cells was as follows: LxPit-1 = 1.8 ± 0.3-fold, LxGR = 1.6 ± 0.3-fold, LxGR + dexamethasone = 9.4 ± 1.7-fold, and LxSp1 = 14.1 ± 0.9-fold. Right panel, each experiment with Me(2)SO-treated P19 cells represents an independent differentiation event.***, p < 0.0001 versus empty vector control (Student's t test). Activation of basal activity by LexA chimeras was as follows: LxPit-1 = 2.1 ± 0.3-fold, LxGR = 2.3 ± 0.3-fold, LxGR + dexamethasone = 7.6 ± 1.1-fold, and LxSp1 = 10.5 ± 0.6-fold. D, responsiveness of LexA fusion constructs to DA in pancreatic RIN and InR1-G9 cells. Values represent the means ± S.E. of three or more independent experiments. Left panel, Mann-Whitney test.**, p < 0.02; *, p < 0.03 versus empty vector control. Activation of basal activity by LexA fusions was as follows: LxPit-1 = 2.1 ± 0.3-fold, LxGR = 2.3 ± 0.6-fold, LxGR + dexamethasone = 8.9 ± 1.1-fold, LxRel = 22.2 ± 1.3-fold, and LxSp1 = 15.7 ± 0.8-fold. Right panel, Student's t test.**, p < 0.008 versus empty vector control. Activation of basal activity by LexA fusions was as follows: LxPit-1 = 3.6 ± 1.0-fold, LxRel = 15.9 ± 2.7-fold, and LxSp1 = 12.3 ± 2.2-fold.



A DA response was not detected in control COS-7 cells transfected with only the D2 receptor expression vector and the 2xLex reporter (Fig. 2B). However, in cells cotransfected with the LxPit-1 expression vector, basal luciferase activity was enhanced 6-fold, and the addition of DA caused a 1.7-fold stimulation. This stimulatory effect was quantitatively similar to that observed with full-length Pit-1 and the PRL promoter in COS-7 cells (Fig. 1). In contrast to LxPit-1, each of the other chimeric factors (i.e. LxGR, LxRel, and LxSp1) elevated basal transcription of the reporter construct, but none was able to mediate a DA response. Because the LxGR construct contains the glucocorticoid receptor ligand-binding domain, full transcriptional function is dependent on the presence of glucocorticoids(19) . Accordingly, dexamethasone activated LxGR-dependent transcription 7-8-fold (see legend to Fig. 2B); however, no further changes in LxGR function were seen upon treatment with DA (Fig. 2B). In the same experiments, LxPit-1 activity was unaffected by the addition of dexamethasone (data not shown).

In P19 cells, full-length Pit-1 can mediate a transcriptional response to DA only once cell differentiation has occurred. As shown in Fig. 2C, the chimeric factor LxPit-1 exhibited a similar pattern of regulation, in which no DA response was detected in undifferentiated P19 cells (leftpanel), but a 50% increase was seen following Me(2)SO induction (rightpanel). LxSp1 and LxGR (with or without dexamethasone) were unable to mediate a response, as noted in COS-7 cells. Moreover, basal luciferase levels were activated equally by LxPit-1 in undifferentiated and Me(2)SO-induced P19 cultures (see legend to Fig. 2C), indicating that DA regulation is not strictly proportional to the transactivating potential of LxPit-1. These data demonstrate the specificity of Pit-1 N-terminal sequences in mediating regulation by DA. Interestingly, in two preliminary experiments (data not shown), retinoic acid-induced P19 cells exhibited negative LxPit-1-dependent regulation by DA (approximately 20% inhibition), further demonstrating that the cell type-specific pattern of regulation is determined by Pit-1 N-terminal sequences.

Consistent with its function in COS-7 and P19 cells, the LxPit-1 chimera mimicked the regulatory pattern of Pit-1 in DA-treated RIN and InR1-G9 cells. As shown in Fig. 2D (leftpanel), LxPit-1 mediated a 40-50% decrease in Lex sitedependent reporter activity in RIN cells, which is quantitatively comparable to the DA effect in cells cotransfected with -422P/luciferase and Pit-1 (Fig. 1). A negative response similar to that of RIN cells was seen in InR1-G9 cells expressing LxPit-1 (Fig. 2D, rightpanel). In both cell lines, transcriptional activation by LxGR or LxSp1 was unaffected by DA. A modest response to DA was observed with LxRel specifically in RIN cells.

G Mutants Specifically Inhibit Transactivation by LxPit-1-(8-80)

Pit-1 expression in RIN cells facilitates inhibition, rather than stimulation, of the PRL promoter by DA. Certain key signaling components that are common to PRL-secreting cells and pancreatic endocrine cells may transduce the inhibitory response. Such components might include not only potential effector proteins (e.g. voltage-dependent Ca channels) as discussed above, but also the heterotrimeric G proteins required for D2 receptor/effector coupling. Indeed, the repertoire as well as the relative levels of pertussis toxin-sensitive G subtypes in RIN cells are remarkably similar to those in GH4 cells(43) , providing a basis for similar D2 receptor coupling in these two cell types.

Activated mutants of G subtypes that interact with lactotrophs (32) and GH4 cells (44) D2 receptors can mimic the inhibitory actions of DA on the PRL promoter(17) . Transfection of the same mutants (i.e. G and G) into RIN cells suppressed the activity of a Pit-1-activated PRL promoter by 25-30% (Fig. 3). To determine if N-terminal sequences of Pit-1 were sufficient to mediate regulation, G expression vectors were cotransfected with the LxPit-1 construct and the 2xLex reporter. G and G mutants inhibited LxPit-1-activated luciferase expression to the same extent as with a full-length Pit-1 (Fig. 3). Control transfections with the LxSp1 construct demonstrated that regulation was dependent on Pit-1 residues 8-80.


Figure 3: LxPit-1-dependent inhibition by activated G mutants in RIN cells. Cells were electroporated with the D2 receptor expression vector (5 µg), either the -422P/luciferase or 2xLexA/luciferase reporter (10 µg), and expression vectors for Pit-1 (5 µg), LxPit-1 (5 µg), LxSp1 (5 µg), G (2.5 µg), or G (2.5 µg) or the appropriate empty vector controls as shown. Values represent the means ± S.D. of three independent experiments.***, p < 0.008 (Mann-Whitney test). Activation of basal activity by the addition of Pit-1 (2.1 ± 0.7-fold), LxPit-1 (1.8 ± 0.8-fold), and LxSp1 (13.9 ± 3.6-fold) was as indicated.



Modularity of the N-terminal DA-responsive Domain of Pit-1

The importance of the Pit-1 POU domain in specifying DNA contacts and protein interactions is well documented. By comparison, little is known about the structure of the N-terminal transactivation domain, how it interacts with the transcription initiation complex, or how it is regulated. Studies using chimeric Pit-1 constructs have indicated that the N-terminal transactivation domain is modular, functioning independently of the C-terminal POU domain(3, 4) . As shown in Fig. 2and Fig. 3, regulation of Pit-1-dependent transactivation by D2 receptor/G protein-mediated pathways can likewise occur in the absence of POU sequences. These data highlight the complexity of hormone-regulated Pit-1 function, demonstrating that the transactivation domain as well as the POU domain (41) can serve as targets for extracellular signals.

In summary, our study demonstrates that Pit-1 can function as a terminal second messenger for inhibitory G protein signals and that the transactivation domain is important in this response. The selective ability of Pit-1 N-terminal sequences to transfer DA regulation to LexA operator sites shows that neither the Pit-1 POU domain nor the Pit-1 DNA-binding site is entirely necessary for DA responsiveness. Protein/protein interactions involving the Pit-1 N-terminal domain are likely to be essential for DA regulation. It remains to be determined whether inhibition of transactivation requires direct modification of Pit-1 residues within the 72-amino acid sequence or an indirect mechanism, such as recruitment or dissociation of a DA-regulated accessory protein. Finally, the ability of LxPit-1 to respond to hormone signals in a number of different cell types indicates that factors associated with the transcriptional regulatory mechanism are not restricted to the pituitary.


FOOTNOTES

*
This work was supported in part by the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a studentship from the Ontario Graduate Scholarship Program.

To whom correspondence should be addressed: Dept. of Clinical Biochemistry, University of Toronto, 100 College St., Toronto, Ontario M5G 1L5, Canada. Tel.: 416-978-8782; Fax: 416-978-4108.

(^1)
The abbreviations used are: PRL, prolactin; DA, dopamine; RSV, Rous sarcoma virus; TRH, thyrotropin-releasing hormone.

(^2)
H. Van Tol, unpublished data.


ACKNOWLEDGEMENTS

We thank Drs. Holly Ingraham, Geof Rosenfeld, Tom Gilmore, and Keith Yamamoto for plasmids used in these studies; Dr. Paul Hamel for advice on P19 cell studies; and Dr. Hubert Van Tol for ligand binding analysis of D2 receptor-transfected cells.

Note Added in Proof-Recent analysis of Pit-1 mutants has shown that phosphorylation at Ser-115 and Thr-220 is not a requirement for Pit-1-mediated signaling in hormone-stimulated cells (Okimura, Y., Howard, P. W., and Maurer, R. A.(1994) Mol. Endrocrinol.8, 1559-1565; Fischberg, D. J., Chen, X.-h., and Bancroft, C.(1994) Mol. Endocrinol.8, 1566-1573). Interestingly, Okimura et al., and Howard and Maurer (Howard, P. W., and Maurer, R. A.(1994) J. Biol. Chem.269, 28662-28669) reported that a chimeric factor containing full-length Pit-1 fused to a GAL4 DNA-binding domain was unable to mediate responses to cAMP or TRH, respectively. Whether differences in hormone responses of GAL4-Pit-1 and LexA-Pit-1-(8-80) reflect differences in signaling pathways, heterologous DNA-binding domains, or DNA recognition sites or are related to the length of the chimeric Pit-1 sequence needs to be explored.


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