Palindromic cAMP Response Element Research Articles

Increased cAMP Levels Modulate Transforming Growth Factor-β/Smad-induced Expression of Extracellular Matrix Components and Other Key Fibroblast Effector Functions

cAMP is a key messenger of many hormones and neuropeptides, some of which modulate the composition of extracellular matrix. Treatment of human dermal fibroblasts with dibutyryl cyclic AMP and forskolin antagonized the inductive effects of transforming growth factor-beta (TGF-beta) on the expression of collagen, connective tissue growth factor, tissue inhibitor of matrix metalloproteinase-1, and plasminogen activator inhibitor type I, four prototypical TGF-beta-responsive genes. Increased intracellular cAMP prevented TGF-beta-induced Smad-specific gene transactivation, although TGF-beta-mediated Smad phosphorylation and nuclear translocation remained unaffected. However, increased cAMP levels abolished TGF-beta-induced interaction of Smad3 with its transcriptional co-activator cAMP-response element-binding protein (CREB)-binding protein (CBP)/p300. Overexpression of the transcriptional co-activator CBP/p300 rescued Smad-specific gene transcription in the presence of cAMP suggesting that sequestration of limited amounts of CBP/p300 by the activated cAMP/CREB pathway is the molecular basis of this inhibitory effect. These findings were extended by two functional assays. Increased intracellular cAMP levels suppressed the inductive activity of TGF-beta to contract mechanically unloaded collagen lattices and resulted in an attenuation of fibroblast migration of mechanically induced cell layer wounds. Of note, cAMP and TGF-beta synergistically induced hyaluronan synthase 2 (HAS2) expression and hyaluronan secretion, presumably via putative CREB-binding sites adjacent to Smad-binding sites within the HAS2 promoter. Our findings identify the cAMP pathway as a potent but differential and promoter-specific regulator of TGF-beta-mediated effects involved in extracellular matrix homeostasis.

Platelet-activating Factor Mediates MMP-2 Expression and Activation via Phosphorylation of cAMP-response Element-binding Protein and Contributes to Melanoma Metastasis

Overexpression of cAMP-response element (CRE)-binding protein (CREB) and activating transcription factor (ATF) 1 contributes to melanoma progression and metastasis at least in part by promoting tumor cell survival and stimulating matrix metalloproteinase (MMP) 2 expression. However, little is known about the regulation of CREB and ATF-1 activities and their phosphorylation within the tumor microenvironment. We analyzed the effect of platelet-activating factor (PAF), a potent phospholipid mediator of inflammation, for its ability to activate CREB and ATF-1 in eight cultured human melanoma cell lines, and we found that PAF receptor (PAFR) was expressed in all eight lines. In metastatic melanoma cell lines, PAF induced CREB and ATF-1 phosphorylation via a PAFR-mediated signal transduction mechanism that required pertussis toxin-insensitive Galphaq protein and adenylate cyclase activity and was antagonized by a cAMP-dependent protein kinase A and p38 MAPK inhibitors. Addition of PAF to metastatic A375SM cells stimulated CRE-dependent transcription, as observed in a luciferase reporter assay, without increasing the CRE DNA binding capacity of CREB. Furthermore, PAF stimulated the gelatinase activity of MMP-2 by activating transcription and MMP-2 expression. MMP-2 activation correlated with the PAF-induced increase in the expression of an MMP-2 activator, membrane type 1 MMP. PAF-induced expression of pro-MMP-2 was causally related to PAF-induced CREB and ATF-1 phosphorylation; it was prevented by PAFR antagonist and inhibitors of p38 MAPK and protein kinase A and was abrogated upon quenching of CREB and ATF-1 activities by forced overexpression of a dominant-negative form of CREB. PAF-induced MMP-2 activation was also down-regulated by p38 MAPK and protein kinase A inhibitors. Finally, PAFR antagonist PCA4248 inhibited the development of A375SM lung metastasis in nude mice. This result indicated that PAF acts as a promoter of melanoma metastasis in vivo. We proposed that metastatic melanoma cells overexpressing CREB/ATF-1 are better equipped than nonmetastatic cells to respond to PAF within the tumor microenvironment.

Modular structure of cAMP response element binding protein 2 (CREB2)

The transcription factor cAMP-response element binding protein 2 (CREB2), a member of the family of basic region leucine zipper proteins, has been suggested to function in the brain as a repressor of long-term memory. Using recombinant proteins we show that CREB2 binds in vitro to the palindromic cAMP response element derived from the secretogranin II gene. Recent studies of the chromogranin B, secretogranin II and enkephalin genes showed that CREB2 functioned as a repressor of cAMP-induced transcription. We analyzed the ability of CREB2 to repress transcription using model promoters. A molecular dissection of the CREB2 molecule revealed that CREB2 lacks a transferable repressor domain suggesting that CREB2 may function solely as a “passive” transcriptional repressor. In contrast, “active” repressor domains derived from the thyroid hormone receptor α or the NK10 zinc finger protein containing a “Krüppel associated box” could be transfered to a heterologous DNA-binding domain and functioned as fusion proteins in repressing transcription of a reporter gene. In addition, a strong activation domain located at the N-terminus was identified in the CREB2 protein suggesting that CREB2 may act as an activator of transcription by binding to different genetic regulatory elements.

The decidualizing effect of progesterone may involve direct transcriptional activation of corticotrophin-releasing hormone from human endometrial stromal cells.

The hypothalamic neuropeptide corticotrophin-releasing hormone (CRH) is also produced by human endometrial cells and is directly involved in the decidualization process as a paracrine inducer. The aim of the present work was to examine the effect of progesterone, the main decidualizing factor, on endometrial CRH, in primary cultures of human endometrial stromal cells. The effect of progesterone was examined by measuring the effects of medroxyprogesterone acetate (MPA) on (i) the concentration of immunoreactive CRH in isolated human endometrial stromal cells and (ii) the activity of the CRH promoter in human endometrial stromal cells transfected with a 0.9 kb fragment of the 5'-flanking region of the human CRH gene coupled to luciferase reporter. The data show that MPA increased the production and secretion of immunoreactive CRH from stromal cells and induced the activity of the CRH promoter, both in a dose-dependent manner. These effects were partially reversed by a molar excess of the antiprogestin RU 486 and were completely abolished in the presence of 100 nmol/l of the cAMP inhibitor, Rp-cAMP. The effect of progesterone on the CRH promoter requires the existence of an intact CRH sequence since experiments carried out with a deleted palindromic cAMP response element (CRE: 5'-TGACGTCA) at -224 bp of the CRH promoter resulted in a complete loss of MPA effect. In conclusion, these data provide evidence that progesterone induces the transcription of CRH gene in human endometrial stroma. This effect coupled with the decidualizing properties of progesterone and CRH may indicate that progesterone and CRH form a decidualizing local pathway within the human endometrium.

Transcriptional regulation by cyclic AMP is essential for development, reproduction and survival: lessons from the transgenic mice.

A great number of hormones act at the cellular level by stimulation of the cAMP signalling pathway, after binding to their specific G protein-coupled transmembrane receptor. Alterations of the cAMP pathway have been implicated in several endocrine diseases such as acromegaly, toxic thyroid adenomas or Cushing’s syndrome. One major cellular effect of the cAMP cascade activation is stimulation of transcription after phosphorylation of nuclear factors by the cAMP-dependent kinase, PKA (Fig. 1). A conserved palindromic cAMP response element (CRE) has been identified in the promoter of various genes regulated by cAMP (i.e. somatostatin, vasoactive intestinal polypeptide, alpha chorionic gonadotrophin, enkephalin, fos, parathyroid hormone (PTH) .... genes). Three proteins have been identified that bind the CRE and are phosphorylated by PKA: CREB (CRE binding protein), CREM (CRE modulator) and ATF-1 (activating transcription factor 1) (1). While CREB and ATF-1 stimulate transcription, some isoforms of CREM can negatively regulate CRE activity. A coactivator of CREB that binds, specifically, CREB phosphorylated by PKA in response to cAMP stimulation, has been cloned and termed CREB binding protein (CBP). CBP appears to be a general transcriptional coactivator for numerous signalling pathways. To evaluate the physiological significance of the various components regulating the CRE, a number of transgenic models have been reported and show the major role of cAMP regulated transcription. CREB null mice generated by homologous recombination die just after birth from respiratory distress (2). Analysis of these animals show that they are smaller than the controls and that they have central nervous system alterations (corpus callosum and anterior commissures reduction). It has been observed previously that mice lacking two of the three main splice product isoforms of CREB have a deficient long-term memory (3). It should be noted that in both models of CREB knockout mice (i.e. the partial and the complete models), an overexpression of CREM and ATF-1 is observed. This suggests a compensatory mechanism between the members of the CREB/CREM/ ATF-1 family. The CREB -/- mice also have impaired T cell development. Interestingly, a T cell proliferative defect and a decreased interleukin-2 production are also observed in transgenic mice expressing a dominantnegative mutant of CREB under the control of the thymocyte-specific CD2 promoter (as the use of the CD2 promoter in the construction of the transgene allows specific expression of the mutant in the thymocytes) (4). In this mutant, the PKA phosphorylation site is mutated and therefore the cAMP activation of CREB is abolished. The same approach has demonstrated the major role of CREB in the development of the pituitary somatotroph. Transgenic mice expressing a similar dominantnegative mutant of CREB under the control of the growth hormone (GH) promoter are dwarf and present with pituitary hypoplasia due to a dramatic reduction in the number of GH cells (5). Targeting a dominantnegative mutant of CREB in the heart using the myocyte-specific alpha-MHC promoter leads to dilated cardiomyopathy in transgenic mice (6). Disruption of the CREM gene by homologous recombination leads to infertility in male mice due to deficient spermiogenesis (7, 8), without hormonal alterations of the gonadotroph axis. Mice homozygous for a targeted mutation of the coactivator CBP die during embryonic development and show neural tube defects (9). Numerous transgenic mice models for the study of transcription regulation by cAMP reported to date have given insights into the major developmental role of the various CRE binding proteins and of CBP. Together with the knockout models, the use of dominant-negative mutants targeted to a given tissue allows the identification of the specific role of CRE binding proteins in each tissue and reveals tissue-specific differences for proteins that seem quite ubiquitously expressed. The CREB null mice suggest that compensation occurs in the CRE binding protein family, in keeping with the essential role of these transcription factors. Nevertheless, a specific role for each member of this family does exist. In this view the development of the ATF-1 null mice and of the double knockout mice will help to explain the function of each nuclear component of the cAMP-regulated transcription (2). The use of the conditional knockout technology which allows control of the timing and/or

CAMP responsiveness of the bovine calpastatin gene promoter

Previous studies have shown that transcription of the gene encoding bovine calpastatin, an inhibitor of the calcium-activated cysteine protease calpain, is upregulated following activation of cAMP-dependent signaling pathways. In this study, deletion and site-directed mutagenesis experiments were performed to identify cis elements conferring cAMP responsiveness. Heterologous promoter assays demonstrated that all cAMP-responsive cis elements were located within −102 nucleotides (nt) of transcription initiation. Deletion of an element (GTCA) at nt +13 that is identical to half of the palindromic cAMP-responsive element (TGACGTCA) identified in other cAMP-responsive gene promoters had no effect on the response of the calpastatin promoter to dibutyryl-cAMP, although a 67% reduction in basal promoter activity was observed. In contrast, two point mutations in a cis element at nt −76 (GTCA to aTCt) abolished cAMP responsiveness. These results demonstrate that the calpastatin promoter sequence between nt −1653 and +130 contains a single cAMP-responsive element (GTCA) located at nt −76, and suggest a direct molecular pathway by which activation of cAMP signaling could lead to increased calpastatin gene transcription and reduction in calpain-mediated proteolysis.

A CRE-like sequence that binds CREB and contributes to cAMP-dependent regulation of the proximal promoter of the human aromatase P450 ( CYP19) gene

The major physiological regulator of human aromatase P450 gene expression in the ovary is follicle stimulating hormone (FSH), which acts by increasing intracellular cAMP levels. This study describes the identification of an element in the aromatase proximal promoter that is critical for the full transcriptional response of this promoter to cAMP. The cAMP-responsive element (CRE)-like sequence (CLS) was originally identified by its sequence similarity to a palindromic CRE, from which it differs by the insertion of a single cytosine. Mutation of the CLS in the context of 278 bp of 5′-flanking DNA resulted in the loss of cAMP-induced reporter gene expression in transfected ovarian luteal cells. A cell line survey EMSA revealed that CLS binding factors are ubiquitously distributed, although the migration pattern of CLS-nuclear protein complexes varied among different nuclear extracts. An extended half-site for binding members of the basic-leucine zipper class of transcription factors was found to be responsible for ovarian luteal cell nuclear protein binding and cAMP-dependent transcriptional transactivation. Competition and supershift EMSAs revealed that the CLS-nuclear protein complexes that regulate cAMP-induced transcription were indistinguishable from homodimeric CREB bound to the CRE oligonucleotide, yet the interaction with the CLS was of lower affinity.

A CRE-like element plays an essential role in cAMP regulation of human SP-A2 gene in alveolar type II cells.

The human has two genes encoding surfactant protein-A (SP-A), termed SP-A1 and SP-A2; the SP-A2 gene is more highly regulated by cAMP and during fetal development than is SP-A1. In this study, by use of primary cultures of human type II cells transfected with fusion genes containing various amounts of SP-A2 5'-flanking DNA linked to human growth hormone (hGH) structural gene, as reporter, we found that -296 bp of SP-A2 upstream sequence is sufficient to direct high basal and cAMP-inducible expression in type II cells, but not in other cell types. By use of competitive EMSA, we observed that nuclear proteins isolated from midtrimester human fetal lung tissue bind specifically to a cAMP response element (CRE)-like sequence, TGACCTTA, at -242 bp, which we have termed CRESP-A2. Binding activity of CRESP-A2 for nuclear proteins from human fetal lung tissue before culture was manifest as two complexes of different mobilities and equivalent intensity. By contrast, upon differentiation of the human fetal lung in culture in the presence of dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), the higher mobility complex was decreased to undetectable levels. By UV cross-linking analysis, using nuclear extracts from midgestation human fetal lung before culture and radiolabeled CRESP-A2 as a probe, we observed binding of proteins of approximately 50, 36, and 30 kDa. When nuclear extracts from human fetal lung cultured in the presence of DBcAMP were analyzed, binding of only the 50- and 36-kDa proteins was apparent. On the other hand, when the canonical CRE (TGACGTCA) known to bind the transcription factor CREB (M(r) approximately 43,000) was used as a probe, binding of only a approximately 43-kDa protein was evident using nuclear extracts from human fetal lung before and after culture. In type II cells transfected with an SP-A2(-296):hGH fusion gene in which CRESP-A2 was mutated, there was a marked reduction of basal and cAMP-stimulated fusion gene expression. These findings indicate that CRESP-A2 serves an important role in mediating basal and cAMP-inducible expression of the human SP-A2 gene in type II cells, that the fetal lung nuclear proteins bound to CRESP-A2 differ from those bound to the canonical palindromic CRE, and that changes in the complex of nuclear proteins bound to CRESP-A2 accompany induction of SP-A gene expression.

Characterization of the cyclic adenosine 3',5'-monophosphate response element of the rabbit surfactant protein-A gene: evidence for transactivators distinct from CREB/ATF family members.

Surfactant protein-A (SP-A) gene transcription in fetal lung explants is stimulated by factors that increase intracellular cAMP. In transfected type II cells, expression of fusion genes containing 991 bp of 5'-flanking DNA from the rabbit SP-A gene linked to the human GH gene as reporter is stimulated more than 20-fold by cAMP. Mutagenesis of a putative cAMP responsive element (CRE) located -261 bp upstream of the SP-A transcription initiation site to a sequence known not to bind the transcription factor CRE-binding protein (CREB) caused a marked decrease in basal and cAMP-inducible reporter gene expression. This element, termed CREsp-a (TGACCTCA), differs by one nucleotide from a palindromic CRE (CREpal, TGACGTCA), which is known to bind CREB as a homodimer. In the present study, we found that mutagenesis of CREsp-a to CREpal also caused a marked decrease in basal and cAMP-induced fusion gene expression. The findings of competitive electrophoretic mobility shift assays (EMSA) using fetal rabbit lung nuclear extracts suggest that different protein complexes bind CREsp-a and CREpal. By UV cross-linking analysis, an approximately 43-kilodalton protein complex was found to interact both with CREsp-a and CREpal; however, purified CREB was ineffective in binding CREsp-a but did bind CREpal. In EMSA using fetal rabbit lung nuclear proteins, antibodies directed against CREB, CRE modulator (CREM), and activating transcription factor-1 (ATF-1) failed to supershift the complex of proteins bound to CREsp-a; whereas, a supershift was evident using CREpal as a probe. Moreover, in competition EMSA using radiolabeled CREsp-a and fetal rabbit lung nuclear proteins, a purified basic leucine zipper (bLZ) polypeptide failed to compete for binding. By contrast, the bLZ polypeptide competed effectively with CREpal for lung nuclear protein binding. This finding suggests that leucine zipper transcription factors do not bind CREsp-a. Additionally, expression of a CREsp-a:HIS3 fusion gene in yeast was unaffected either by CREB or bLZ polypeptides fused to the GAL4 activation domain. By contrast, HIS3 expression was markedly induced both by CREB and bLZ fusion proteins in a CREpal:HIS3 yeast strain. By competition EMSA using mutagenized CREsp-a oligonucleotides, the critical protein-binding nucleotides in CREsp-a were found to constitute a hexameric element, TGACCT, which corresponds to a binding site for members of the steroid receptor superfamily. Since the TGACCT motif is present in the SP-A gene as a single site, we propose that a unique orphan member of the steroid receptor superfamily may bind to this element.

Transcription of the Rat Glucagon Gene by the Cyclic AMP Response Element-Binding Protein CREB Is Modulated by Adjacent CREB-Associated Proteins

The cyclic AMP (cAMP) response element (CRE) of the rat glucagon gene (Glu-CRE, 5'-TGACGTCA-3') mediates transcriptional responses to 8-bromo-cAMP and protein kinase A (PKA) in a glucagon-producing hamster islet cell line (InR1G9). By several different DNA-protein binding assays, we show that the transcription factor CREB binds to the CRE octamer and that additional nuclear proteins bind to sequences adjacent to the CRE. Mutation of the Glu-CRE octamer attenuates both the binding of CREB and cAMP-dependent PKA-stimulated transcriptional activity in transient transfection experiments but does not affect the binding of adjacent CREB-associated proteins. Progressive deletions and clustered point mutations of the sequences flanking the Glu-CRE identify sequences (5'-TCATT-3') located both 5' and 3' to the core CRE octamer that bind several proteins. Two proteins with molecular masses of 80 and 100 kDa bind to each of the 5' and 3' TCATT sites. Formation of additional protein-DNA complexes containing 45- and 20-kDa proteins depends upon the integrity of both TCATT sequences. Deletion or point mutation of the TCATT motif located on the 3' side of the CRE octamer results in enhanced transcriptional responses to PKA, suggesting that the CREB-associated proteins decrease the ability of CREB to mediate PKA-stimulated transcription. Results from these studies demonstrate that nucleotides flanking the core CRE octamer can influence the activity of the CRE by serving as binding sites for proteins that modulate the function of CREB and suggest a mechanism to explain why some consensus palindromic CREs are less responsive to cAMP stimulation than others.

Transcription of the rat glucagon gene by the cyclic AMP response element-binding protein CREB is modulated by adjacent CREB-associated proteins.

The cyclic AMP (cAMP) response element (CRE) of the rat glucagon gene (Glu-CRE, 5'-TGACGTCA-3') mediates transcriptional responses to 8-bromo-cAMP and protein kinase A (PKA) in a glucagon-producing hamster islet cell line (InR1G9). By several different DNA-protein binding assays, we show that the transcription factor CREB binds to the CRE octamer and that additional nuclear proteins bind to sequences adjacent to the CRE. Mutation of the Glu-CRE octamer attenuates both the binding of CREB and cAMP-dependent PKA-stimulated transcriptional activity in transient transfection experiments but does not affect the binding of adjacent CREB-associated proteins. Progressive deletions and clustered point mutations of the sequences flanking the Glu-CRE identify sequences (5'-TCATT-3') located both 5' and 3' to the core CRE octamer that bind several proteins. Two proteins with molecular masses of 80 and 100 kDa bind to each of the 5' and 3' TCATT sites. Formation of additional protein-DNA complexes containing 45- and 20-kDa proteins depends upon the integrity of both TCATT sequences. Deletion or point mutation of the TCATT motif located on the 3' side of the CRE octamer results in enhanced transcriptional responses to PKA, suggesting that the CREB-associated proteins decrease the ability of CREB to mediate PKA-stimulated transcription. Results from these studies demonstrate that nucleotides flanking the core CRE octamer can influence the activity of the CRE by serving as binding sites for proteins that modulate the function of CREB and suggest a mechanism to explain why some consensus palindromic CREs are less responsive to cAMP stimulation than others.

A novel cAMP-dependent regulatory region including a sequence like the cAMP-responsive element, far upstream of the human CYP21A2 gene

Deletion mutants in the 5' upstream sequence of the human CYP21A2 gene demonstrated a novel regulatory DNA element responsible for cAMP-dependent expression of the gene in the transient expression system using Y-1 cells (mouse adrenocortical tumor cell line). This regulatory element (-2574 to -2489 bp) was also found to exhibit a strong enhancer activity through heterologous promoters in response to cAMP and to contain a sequence like the cAMP-responsive element (CRE) and a CAAT-like sequence. The CRE-like sequence has a five-base motif (5'-TGACG-3') of the palindromic CRE consensus (TGACGTCA). Competitive gel mobility shift assays using nuclear extracts of bovine adrenal cortex with sequences typical of the binding sites for the binding proteins of CRE and the CAAT-like sequence revealed that these binding proteins, or related factors, bound to their cognate DNA binding sites in the upstream enhancer region of the CYP21A2 gene. These two enhancer elements and their cognate binding factors cooperate with previously identified tissue-specific enhancers (adrenal-specific protein factor and Ad4-like sequences) and their binding factors to express a high level of cAMP-responsive expression of the CYP21A2 gene.

Transcriptional regulation of inhibin beta B messenger ribonucleic acid levels in TM.4 or primary rat Sertoli cells by 8-bromo-cyclic adenosine monophosphate.

FSH, a major regulator of inhibin production in the testis, is believed to exert its effects via cAMP second messenger system. Inhibin alpha-subunit gene appears to be regulated by cAMP and has a palindromic cAMP response element sequence TGACGTCA. However, the regulation of the inhibin beta B-subunit gene by cAMP has been less clear. It has been assumed that beta B may not be regulated by cAMP, based mainly on observations that FSH stimulates only alpha, not beta B, mRNA levels, and that the 5'-up-stream regulatory region of the beta B gene does not contain the classical cAMP response element. However, we have observed that 8-bromo-cAMP stimulates beta B mRNA levels in both primary Sertoli (approximately 2-fold) and TM.4 cells (approximately 5-fold). We examined whether this cAMP-induced increase in beta B mRNA levels is the result of increased transcription or altered mRNA stability. Data from nuclear run-on assays demonstrate about a 2-fold increase in relative mRNA synthesis rates in primary Sertoli-cells and about a 4- to 5-fold increase in TM.4 cells. Transfection studies in TM.4 and JEG.3 cell lines with beta B:luciferase chimeric reporter gene constructs containing 1.5 kilobases of the beta B 5'-up-stream regulatory region revealed marked cAMP induction of reporter gene activity in both cell types.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding constant determination studies utilizing recombinant delta CREB protein.

In this study, we report the binding constants (Kd) of the cAMP-responsive element binding protein (delta-CREB) for various cAMP-response element (CRE) motifs. We utilized purified recombinant delta CREB protein in binding reactions with natural CRE motifs found in the promoter of two neuropeptide hormone genes and with several variant CRE motifs. The Kd of delta CREB for the perfectly palindromic CRE, TGACG-TCA, found within the somatostatin promoter is estimated to be 5.0 x 10(-9) M. The Kd of delta CREB for the variant CRE motif TG_CGTCA found within the enkephalin promoter is calculated to be in the 3 x 10(-8) M. These studies provide an in vitro quantitative assessment of the binding affinity of delta CREB for various CRE motifs.

Binding Specificity of Cyclic Adenosine 3′,5′-Monophosphate- Responsive Element (CRE)-Binding Proteins and Activating Transcription Factors to Naturally Occurring CRE Sequence Variants

The human glycoprotein hormone alpha gene contains a palindromic cAMP-responsive element (CRE), TGACGTCA, which is critical for both basal and cAMP-stimulated expression. Members of the CRE-binding protein (CREB)/activating transcription factor transcription family as well as members of the Jun/Fos family have been shown to bind to this sequence. In nonprimate species, the alpha gene contains a variant of the CRE sequence TGAtGTCA, which differs by a single base substitution. The functional properties of promoters containing variant CREs were examined in transient expression assays in JEG-3 cells (human placental cells) and GH3 cells (rat pituitary cells). Relative to the palindromic CRE, the basal level of expression was reduced with the variant CRE constructions. However, 8-bromo-cAMP stimulated the expression of constructions containing either the palindromic or the variant CREs. Protein interactions with the palindromic and variant CREs were also examined. By Southwestern blot analyses in which proteins were initially separated on a denaturing sodium dodecyl sulfate-gel, transferred to a membrane, and then probed with radio-labeled DNA, several different proteins were bound by either the palindromic or the variant CRE sequences. However, the binding of the variant CRE to CREB was markedly reduced. In nondenaturing gel shift assays, multiple protein-DNA complexes were also formed with both types of CREs, but the variant CRE lacked a major complex that may correspond to homodimers of CREB. To determine the protein composition of these complexes, the gel shift bands were eluted and subjected to Southwestern blot analysis. CREB was identified as one of the protein components in several of the gel shift complexes formed with the variant CRE. We conclude that although the variant CRE binds CREB homodimers poorly, CREB can still interact with this element when associated with other proteins. Thus, variants of the palindromic CRE sequence may provide a mechanism for specifying interactions with different combinations of CREBs.