An Additional Region of Coactivator GRIP1 Required for Interaction with the Hormone-binding Domains of a Subset of Nuclear Receptors*

Transcriptional coactivators of the p160 family (SRC-1, GRIP1, and p/CIP) associate with DNA-bound nuclear receptors (NRs) and help the NRs to recruit an active transcription initiation complex to the promoters of target genes. Previous studies have demonstrated the importance of the NR interaction domain (NID) of p160 proteins containing three NR box motifs (LXXLL) for the interaction with the hormone-binding domains of NRs. Here we report that, in addition to NID, another region of coactivator GRIP1 (amino acids 1011–1121), called the auxiliary NID (NIDaux), is requiredin vitro and in vivo for efficient interaction with a subset of NRs, including the glucocorticoid receptor (GR), androgen receptor, and retinoic acid receptor α. A second group of NRs, which includes the progesterone receptor, retinoid X receptor α, thyroid hormone receptor β1, and vitamin D receptor, required only NID for efficient interaction. For binding to GR, the NID and NIDaux of GRIP1 must act in cis, but deletion of up to 144 amino acids between the two regions did not reduce binding efficiency. Amino acids 1011–1121 of GRIP1 also contain a p300 interaction domain, but mutational analysis indicated that the p300 interaction function within this region is separable from the ability to contribute to GR hormone-binding domain binding. SRC-1 lacks an NIDaux activity equivalent to that in GRIP1.

The nuclear receptor (NR) 1 superfamily is a large group of structurally homologous transcription factors that includes the receptors for the five steroid hormones (Class I receptors); receptors for thyroid hormone, vitamin D, and retinoic acid (Class II receptors); and a group called orphan receptors in which cognate ligands are unknown or unnecessary (1)(2)(3)(4). NRs play critical roles in cellular responsiveness to many types of internal and external signals during development and adult life of all higher eukaryotes. NRs are typically composed of three major functional domains (5-7); many, but not all, NRs have an N-terminal activation domain (AD) called AF-1 that carries no sequence homology between different NRs. A highly conserved DNA-binding domain (DBD) in the central region of the polypeptide chain, composed of two four-cysteine zinc fingers, is the hallmark of the NR family. The C-terminal hormone-binding domain (HBD), which carries a lower degree of sequence homology among diverse NRs, is responsible for hormone binding in Class I and II NRs and also carries a second important transactivation domain called AF-2. Hormone binding controls the biological activity of the Class I and II NRs. NRs regulate the expression of specific genes by interacting directly or indirectly with specific enhancer elements in the promoters of the target genes (2,8,9). The promoter-bound NRs help to open the chromatin structure around the enhancer element (10,11) and also are proposed to help recruit a transcription initiation complex to the promoter by binding to components of the transcription complex (2). Class I NRs bind DNA as homodimers, whereas Class II NRs generally form heterodimers with the retinoid X receptors (3,4).
The most well documented mechanism of gene activation by NRs involves direct binding of an NR dimer, through its DBDs, to specific enhancer elements. In addition, many NRs have been shown to modulate the activities of other types of transcription factors (e.g. AP1) indirectly, by binding to the other factors directly, or perhaps through associated coactivator proteins; this modulation can involve DNA binding by the NR (12) or no DNA binding (8,9,13). In all cases, the ability of the NRs to activate gene transcription depends on the actions of the AF-1 and AF-2 ADs. These two ADs often contribute synergistically to transcriptional activation and probably have different mechanisms of activation. These mechanisms may involve both direct and indirect (through adaptor or coactivator proteins) contacts with components of the basal transcription machinery and chromatin (2,14). Understanding of the mechanism of AF-2 function has been recently enhanced by the discovery of a family of 160-kDa proteins that bind in a hormone-dependent manner to the HBDs of Class I and II NRs and thereby enhance transcriptional activation (14,15). This family consists of three genetically distinct but structurally and functionally related proteins: SRC-1 (steroid receptor coactivator 1) (9, 16), GRIP1 (glucocorticoid receptor-interacting protein 1) (17, 18) (also known as TIF2 (transcriptional intermediary factor 2) (19)), and p/CIP (p300/CBP-interacting protein) (13) (also known as ACTR (activator of the thyroid and retinoic acid receptors) (20), RAC3 (receptor-associated coactivator 3) (21), AIB1 (amplified in breast cancer 1) (22), and TRAM-1 (thyroid hormone receptor activator molecule 1 (23)). Members of the p160 family share ϳ40% sequence identity with regions of high and low homology interspersed across their 1400-amino acid length. These transcriptional coactivators do not enhance the expression of genes by themselves because they do not bind to DNA and are not components of the basal transcription machinery. Rather, they are recruited to the promoters through their contacts with the NRs. The p160 coactivators can bind, and may exist in a complex with, two other types of coactivators: CBP or its homologue p300 and p/CAF (p300/CBP-associated factor) (9,20,24). All three families of coactivators help to mediate the activities of NRs, and all three contain histone acetyltransferase activities that may contribute to their functions as coactivators by locally affecting chromatin conformation (20,(25)(26)(27). CBP can also bind to components of the basal transcription machinery (28).
The ability of p160 coactivators to interact with NR HBDs is essential for their coactivator function (29), which is presumably accomplished by serving as adaptors that link AF-2 AD in NR HBD with components of chromatin and the transcription machinery. Mutational analyses of the AF-2 domains of several NRs have provided a strong correlation between loss of transactivation activity and binding to p160 coactivators (16,18,19). The p160 coactivators interact efficiently with the HBDs of all Class I and II NRs (14,29) and also with some orphan receptors (30). 2 To recognize the diverse group of NR HBDs, the p160 coactivators use multiple mechanisms of interaction (31). Each p160 coactivator molecule contains three leucine-rich motifs (LXXLL, where X is any amino acid), called NR boxes, clustered in the central region of the polypeptide chain (13,29,32,33), called the NR interaction domain (NID). Each NR box motif is capable of binding specifically and in a hormone-dependent manner to a diverse group of NR HBDs, but the NR binding preferences of each NR box are somewhat different (13,29,31,33). Thus, although no single NR box motif has a universal ability to bind all NR HBDs efficiently, the multiple motifs present in the p160 proteins collectively contribute to the broad NR binding specificity.
Here we report an additional mechanism that enables the GRIP1 coactivator to interact efficiently with some of the NR HBDs. We found that GRIP1 NID was not sufficient by itself to support an efficient interaction with some NR HBDs. This led to identification of a new region of GRIP1 required for efficient interaction with a subset of NR HBDs.

EXPERIMENTAL PROCEDURES
Plasmids-The yeast expression vector pGAD424.GRIP1/fl for the fusion protein of Gal4 AD and full-length GRIP1 was described previously (18). The yeast expression vectors for the fusion proteins of Gal4 AD and different GRIP1 fragments were made by subcloning PCRamplified GRIP1 cDNA fragments into the EcoRI/BamHI sites of pGAD424 (CLONTECH), which has a leu2 marker gene, or into pHGAD, which was made by inserting a PCR-amplified fragment (containing the adh1 promoter, Gal4 AD, and adh1 transcription termination signal from pGAD424) into the XhoI/SacI sites of pH2, which has a 2 replication origin and a his3 selection marker and was derived from pRS423 as described (34). The yeast expression vectors for fusion proteins of Gal4 DBD and the HBDs of the mouse glucocorticoid receptor (GR), human androgen receptor (AR), human progesterone receptor (PR), human retinoic acid receptor ␣, human retinoid X receptor ␣, human thyroid receptor ␤1 (TR␤1), and human vitamin D receptor were described previously (17,18,29). The yeast expression vector for the Gal4 DBD-p300 fusion protein was made by inserting a PCR-amplified p300 (35) cDNA fragment (coding for amino acids 1856 -2414) into the SmaI/SalI sites of pGBT9 (CLON-TECH). Internal deletions and point mutations of the GRIP1 sequence were made by four-primer PCR as described previously (36), and the resulting DNA sequences were subcloned into the EcoRI/BamHI sites of pGAD424. The SRC-1-GRIP1 fusion gene was also constructed by fourprimer PCR, and the resulting DNA sequence was subcloned into the SalI/BglII sites of pGAD424. 3 The bacterial expression vectors for the fusion proteins of GST with GRIP1 fragments 563-767 and 563-1121, pGEX-4T1.GRIP1 563-767 His 6 and pGEX-4T1.GRIP1 563-1121 His 6 , were made by inserting PCR-amplified GRIP1 cDNA fragments into the BamHI/XhoI sites of pGEX-4T1.His 6 (31). For in vitro transcription and translation of GR and TR, pSG5.rGR (31) and pSG5.hTR␤1 (37) were described previously.
Yeast Two-hybrid Assays-Yeast two-hybrid assays for the interaction between GRIP1 fragments and NR HBDs were performed as described previously (29). Where indicated, the following hormone concentrations were added to yeast cultures for 14 -16 h before harvest: 10 M deoxycorticosterone for GR, 100 nM dihydrotestosterone for AR, 500 nM progesterone for PR, 10 M all-trans-retinoic acid for the retinoic acid receptor, 10 M 9-cis-retinoic acid for the retinoid X receptor, 10 M 3,5,5Ј-triiodo-L-thyronine for TR, and 1 M 1,25-dihydroxyvitamin D 3 for the vitamin D receptor. ␤-Galactosidase activities are shown as the mean Ϯ S.D. from three independent yeast transformants.
GST-dependent Protein-Protein Interaction Assays-Expression of GST-GRIP1-(563-1121)-His 6 or GST-GRIP1-(563-767)-His 6 in Escherichia coli, purification of fusion proteins, loading of measured amounts of fusion proteins on glutathione-agarose beads, incubation of in vitro synthesized TR and GR with the beads, and analysis of bound TR and GR were performed as described previously (31). Eluted bead-bound proteins and input labeled proteins were subjected to SDS-polyacrylamide gel electrophoresis. The fraction of input NR that was bound to beads was determined by PhosphorImager analysis of the gel. For peptide competition assays, 35 S-labeled in vitro translated GR was incubated with 1.6 M glutathione-agarose-bound GST-GRIP1-(563-767)-His 6 or GST-GRIP1-(563-1121)-His 6 in the presence of increasing amounts of peptide KENALLRYLLDKDD (synthesized by the University of California at San Francisco Biomolecular Resource Center). The rest of the procedure was performed as described above. The amount of bound GR was calculated relative to the amount of retained receptor in the absence of peptide.

RESULTS
The Minimum Fragment of GRIP1 Required to Bind GR HBD-The importance of the three clustered NR box or LXXLL motifs in the NID of the p160 coactivator family for binding to the HBDs of all NRs has been established (13,29,32). However, although short polypeptides containing one or more of the NR box motifs are sufficient for binding some NR HBDs (32), our preliminary experiments had indicated that the requirements for GR HBD binding to GRIP1 were more complex. To define the minimum fragment of GRIP1 required for binding GR HBD, we tested the interactions of GR HBD with various GRIP1 fragments in the yeast two-hybrid system (38). In these assays, GR HBD was expressed as a fusion protein with Gal4 DBD, and GRIP1 fragments were fused with Gal4 AD. Interactions between the HBD and a GRIP1 fragment result in the activation of a ␤-galactosidase reporter gene controlled by a Gal4 enhancer. Among the GRIP1 fragments tested, only GRIP1-(730 -1121) exhibited the ability to interact with GR HBD in the yeast two-hybrid assay (Fig. 1, left panel). Note that GRIP1-(730 -1121) contains only the third of the three LXXLL motifs (NR box III), but it interacted with GR HBD as strongly as full-length GRIP1 (1462 amino acids). We have previously shown that NR box III is the most important NR box for binding GR HBD (29). However, GRIP1-(5-765), which contains all three LXXLL motifs, and GRIP1-(1121-1462), which has no NR box motifs, did not interact with GR HBD. All the tested GRIP1 fragments were expressed and could interact with control proteins in the yeast two-hybrid assays (Fig. 1,  right panel).
A Specific Region of GRIP1 Separate from NID Is Important for the Interaction with Both GR HBD and p300 -Since GRIP1-(730 -1121) exhibited full activity to interact with GR HBD, this fragment was used as a starting point for a further deletion analysis in the yeast two-hybrid assays. Deletion of 20 N-terminal amino acids, including NR box III, resulted in the complete loss of interaction with GR and PR HBDs (Fig. 2,  fragment D); thus, in this context, where NR boxes I and II were missing, NR box III was absolutely required for the interaction of GRIP1-(730 -1121) with NR HBDs. Deletion of amino acids 1020 -1121 or 934 -1121 from GRIP1-(730 -1121) (Fig. 2, fragments B and C) also resulted in dramatically reduced interaction with GR HBD, but had only minor effects on binding of PR HBD. Thus, GRIP1-(730 -1121) is the minimum single fragment of GRIP1 that maintains the ability to interact with GR HBD, although it extends far downstream from NID. In contrast, efficient binding of PR HBD requires NID, but not the auxiliary downstream sequences.
Internal deletions of amino acids 869 -938 (which correspond to a natural splicing variant of GRIP1) 4 or amino acids 868 -1010 did not affect the interaction between GRIP1 and GR or PR HBD (Fig. 2, fragments H and I). Therefore, GRIP1 amino acids 1011-1121, which we have named the auxiliary NID (NIDaux), constitute a separate region required for efficient binding to GR HBD, but not PR HBD. GRIP1-(883-1121), GRIP1-(939 -1121), and GRIP1-(1011-1121) (fragments E-G) alone did not exhibit any ability to bind GR or PR HBD, but they all bound to p300. Thus, NIDaux alone did not bind GR HBD, but functioned in the context of one or more of the upstream NR boxes I-III. GRIP1 amino acids 1011-1121 not only functioned as an auxiliary domain for binding of GR HBD, but also were necessary and sufficient for the interaction with p300 (fragments G and I). p300 was previously shown to bind to this region of GRIP1 and the related p160 coactivators (20,24,29).
To study the mechanism for collaboration between NID and NIDaux in the binding of GR HBD, we tested whether these two regions of GRIP1 must work in cis (present in the same protein fragment) or can work in trans (as separate protein fragments). Efficient binding of GR HBD occurred only when NID and NIDaux were expressed as a fused protein in the yeast two-hybrid system (Fig. 3). All of the GRIP1 fragments in this experiment were functionally expressed since fragments that included amino acids 939 -1121 or 1011-1121 interacted with p300 (Fig. 2), and GRIP1-(730 -868) could interact with PR HBD (data not shown) in the yeast two-hybrid system. . We therefore used the yeast two-hybrid system to test several other Class I and II NR HBDs for their dependence on the NIDaux of GRIP1. Since different NR HBDs exhibit different preferences for the three NR boxes (13,29), we used GRIP1 fragments that included all three NR box motifs of GRIP1 in these experiments. Inclusion of all three LXXLL motifs also presents a more physiological context for the interactions. One GRIP1 fragment, GRIP1-(563-1121), contained NID and NIDaux; the second fragment, GRIP1-(563-1019), contained only NID. GR, AR, and retinoic acid receptor ␣ belong to a subset of NRs that interacted efficiently with GRIP1 NID only when NIDaux was also present; in contrast, PR, retinoid X receptor ␣, TR␤1, and vitamin D receptor belong to another group of NRs that did not require NIDaux for efficient interaction (Fig. 4).
Enhancement of GR Binding in Vitro by NIDaux-The ability of two different GRIP1 fragments to interact with fulllength GR and TR␤1 was tested in vitro. GRIP1-(563-767), which contains NID, and GRIP1-(563-1121), which contains NID and NIDaux, were expressed in E. coli as fusion proteins with GST and purified by affinity chromatography. 35 S-Labeled TR and GR were synthesized in vitro in the presence or absence of the appropriate hormone and incubated with GST-GRIP1 proteins bound to glutathione-agarose beads. Bound material was eluted and analyzed by SDS-polyacrylamide gel electrophoresis and PhosphorImager analysis. TR interacted well with GRIP1-(563-767) and GRIP1-(563-1121) (Fig. 5a), indicating that the NID of GRIP1 is sufficient for efficient interaction with TR. However, GRIP1-(563-767) bound GR poorly when compared with the longer GRIP1-(563-1121); the shorter GRIP1 fragment also bound GR less efficiently than TR, whereas the longer GRIP1 fragment bound GR and TR equally. All interactions were hormone-dependent, and the antagonist RU486 could not substitute for dexamethasone to induce GR-GRIP1 interaction. These data were consistent with our observations in the yeast two-hybrid assays indicating that GRIP1 NID by itself was not sufficient for efficient binding of GR HBD.
To quantify the effect of GRIP1 NIDaux on GR and TR binding, different quantities of the two purified GST-GRIP1 fragments were loaded onto glutathione-agarose beads and incubated with labeled GR or TR. These titration curves demonstrated a difference of 50-fold in the concentrations of the two different GST-GRIP1 fragments required to achieve 50% retention of labeled GR, but only a 7-fold difference for binding TR (Fig. 5b). The results suggest that in this in vitro assay, the shorter GRIP1 fragment binds all NR HBDs less efficiently than the longer GRIP1 fragment; in addition, the fact that the difference is much more pronounced for GR than for TR indicates that efficient GR binding requires NIDaux. In a peptide competition assay, peptide KENALLRYLLDKDD, which is derived from GRIP1 NR box III and its surrounding sequence, inhibited the interaction of GR with GRIP1-(563-767) more efficiently than with GRIP1-(563-1121), indicating a stronger interaction between GR and GRIP1-(563-1121) (data not shown). Thus, the presence of NIDaux dramatically enhanced the affinity of GR binding in vitro and in vivo.
GRIP1 NIDaux Enhances the Ability of SRC-1 NID to Interact with GR and AR HBDs-SRC-1 and GRIP1 exhibit extensive co-linear sequence homology (18); both have a highly conserved central NID that contains three LXXLL motifs. SRC-1a, one of the alternative splicing forms of SRC-1, also contains a fourth LXXLL motif at its extreme C terminus. Binding of GR and AR HBDs by SRC-1a is almost entirely due to NR box IV  (29). Truncation of SRC-1a immediately after the p300-binding domain dramatically reduced its binding to GR and AR HBDs, but did not affect its binding to TR HBD in the yeast two-hybrid system (Fig. 6, SRC-1-(1-977)). The same result was obtained with SRC-1a-(1-1433), which lacked only the last eight amino acids including NR box IV (data not shown). One possible reason for the poor binding of GR and AR HBDs by the NIDs of SRC-1a-(1-977) and SRC-1a-(1-1433) could be the lack of a functional NIDaux, like that in GRIP1. Fusing the NIDaux of GRIP1 (contained in amino acids 750 -1121) to the central NID of SRC-1 (contained in amino acids 1-753) restored the ability to bind GR and AR HBDs. Thus, although SRC-1 lacks a functional counterpart to GRIP1 NIDaux, the GRIP1 NIDaux can cooperate with the SRC-1 NID to restore binding of GR and AR HBDs.
The p300 Binding Activity of GRIP1 Is Genetically Separable from the NIDaux Function-Since the GRIP1-(1011-1121) region is involved in the interaction with both GR HBD and p300, we tested whether the two activities were mechanistically related, e.g. whether GR HBD binding by GRIP1 requires an intact p300 binding activity. Protein sequence comparisons within this region of the three non-allelic members (GRIP1/ AIB1/SRC-1) of the p160 coactivator family identified two conserved segments that partially resemble LXXLL motifs (Fig. 7). These motifs were previously named LCD4 and LCD5 (leucine charged domain) (13). LL-to-AA substitutions in each motif were used to test their potential involvement in GR HBD and/or p300 interactions. In yeast two-hybrid assays, the LCD4  6. Interaction of an SRC-1-GRIP1 fusion protein with NR HBDs. A fusion protein containing the central NID of SRC-1 and the NIDaux of GRIP1 was compared with full-length SRC-1a and a Cterminally truncated form of SRC-1a for binding to three different NR HBDs in yeast two-hybrid assays. Each coactivator protein was fused to Gal4 AD and coexpressed with Gal4 DBD-NR HBD fusion proteins in yeast strain SFY526 in the presence of appropriate hormones. The resulting ␤-galactosidase (␤-Gal) activities are shown. u, units. Numbers above each protein diagram represent SRC-1a amino acids, whereas numbers below each diagram represent GRIP1 amino acids. NR boxes (NRB) are represented by vertical bars. CID, CBP/p300 interaction domain. mutation L1063AϩL1064A (Fig. 7, Mut1) in GRIP1 had no effect on the interaction of GRIP1-(730 -1121) with either GR HBD or p300. In contrast, the LCD5 mutation L1079Aϩ L1080A (Fig. 7, Mut2) resulted in a 70% loss of p300 binding activity, but had no effect on the interaction with GR HBD. Combining the four amino acid substitutions (Fig. 7, Mut1 ϩ Mut2) had effects similar to Mut2 alone. Although these results did not identify the residues of NIDaux required for efficient GR HBD binding, they helped to localize the p300-binding site and demonstrated that the NIDaux and p300 binding functions of GRIP1 are genetically separable. DISCUSSION For GRIP1 and the other p160 coactivators to mediate transcriptional activation by NRs, the NRs must bind the p160 coactivators (13,29,32,33); this interaction is presumably responsible for recruiting the p160 coactivator, and perhaps a complex of associated coactivators including CBP or p300 (9,20,24), to the promoter, where the coactivator complex presumably helps to remodel chromatin and/or to recruit a transcription initiation complex. This NR-coactivator binding is primarily due to interaction of the coactivators with the highly conserved AF-2 domain within the HBDs of most NRs (16,18,19,31,37). Whereas the p160 coactivators can also interact with the AF-1 domains of at least a few steroid receptors (39,40), the binding of these coactivators to the AF-2 domain in the HBDs of NRs is stronger (39) and appears at this time to be universal, at least for all NRs that function as transcriptional activators. This universality is rather remarkable in light of the fact that the degree of amino acid sequence homology among the NR HBDs that interact with p160 coactivators can be as low as 10% (41). Nevertheless, the three-dimensional architecture of the HBD among a diverse group of Class I and II NRs is highly conserved (42)(43)(44)(45)(46). The almost universal interaction between p160 coactivators and NR HBDs suggests that this interaction must depend on highly conserved residues and surface features of the HBD. In fact, a recent mutational analysis (37) and an x-ray crystallographic analysis (31) confirmed that many of the TR␤1 HBD residues that bind GRIP1 are highly conserved among the NR superfamily. These residues are found in ␣-helices 3, 4, 5, and 12. These helices form a hydrophobic groove surrounded by a rim of charged residues on the surface of the HBD; the groove accommodates the core leucine residues of the NR box motif (31). However, although highly conserved residues form the core of the HBD site that interacts with GRIP1, the 10% sequence identity among the HBDs of diverse members of the NR family suggests that p160 coactivators may encounter considerable structural diversity around the core interaction sites of various NR HBDs. In fact, amino acid sequences flanking the LXXLL motif of NR box II interact directly with surface residues from the C-terminal region of the TR HBD that are not conserved among the NR family; alterations in these TR residues affect the NR box binding preference of TR (31).
From previous studies (29) and the one reported here, we now can understand in more detail how the p160 coactivators accomplish the task of interacting with a broad spectrum of NR HBDs with diverse sequences. They do so by using a variety of interaction sites to make contact with the NR HBDs. These include three NR box motifs (or four for SRC-1a) and the auxiliary GRIP1 site described here. Each NR box can bind a broad spectrum of NR HBDs, but interacts inefficiently with a subset of NR HBDs. The subset of NR HBDs bound efficiently is different for each of the NR boxes (29). Interaction affinity and specificity of each NR box depend on the sequence of the core LXXLL motif and on the flanking sequences. The relative importance of core and flanking sequences varies for different NR box-NR HBD combinations (31). Among the cluster of three NR boxes located in the central NID of the p160 polypeptide chain, NR box I appears to play a relatively minor role since, in GRIP1/TIF2, this motif binds weakly to most NR HBDs (29,33). But NR boxes II and III, which are conserved in all three p160 family members, and NR box IV, which is found only at the extreme C terminus of SRC-1a, all contribute unique HBD binding specificities (29). Thus, although no single NR box can bind efficiently to all NR HBDs, the combination of the multiple NR boxes in each p160 coactivator recognizes an extremely broad spectrum of NR HBDs. However, by itself, the NID of GRIP1 or SRC-1 with three NR box motifs still binds some NR HBDs inefficiently, including those of GR, AR, and retinoic acid receptor ␣. GRIP1 and SRC-1 use different strategies to solve this problem. GRIP1 has an auxiliary region (NIDaux), located between amino acids 1011 and 1121, that cannot by itself bind NR HBDs, but cooperates with NID to accomplish efficient binding of GR and AR HBDs. SRC-1 lacks an auxiliary HBD binding function comparable to that found in GRIP1. Instead, SRC-1a contains an additional NR box IV located at the extreme C terminus of the protein, which binds GR and AR HBDs independently of the central NID (NR boxes I-III) (29). Another isoform of SRC-1, called SRC-1e, is produced by alternative splicing of the same transcript; SRC-1e lacks the C-terminal NR box IV (9,47,48) and, as a result, binds GR and AR poorly. 5 How does GRIP1 NIDaux promote better binding of GR and AR HBDs? One possible model is that both NID and NIDaux of GRIP1 make direct contact with GR and AR HBDs. The binding interaction of the HBD with each individual region is weak, 5 H. Ma and M. R. Stallcup, manuscript in preparation. FIG. 7. Within the GRIP1-(1011-1121) region, the auxiliary GR HBD binding activity and p300 binding activity are genetically separable. GRIP1-(730 -1121) with either wild-type (wt) sequence or the indicated mutations was expressed as a fusion protein with Gal4 AD and coexpressed with a fusion protein of Gal4 DBD with either GR HBD or p300 in yeast strain SFY526 in the presence of 10 M deoxycorticosterone (for GR) or in the absence of hormone (for p300). The resulting ␤-galactosidase (␤-Gal) activities are shown. u, units. but the two interactions together provide sufficient binding energy to form a stable complex. In another possible model, GRIP1 NIDaux could function through intramolecular interactions that stabilize a conformation of NID that is favorable for binding GR and AR HBDs. Although our data do not discriminate between these two models, they indicate that NID and NIDaux must function in cis to bind GR HBD and that 144 amino acids between the two GRIP1 regions can be deleted without destroying their ability to cooperate in binding GR HBD. Although GRIP1 NIDaux enhances the affinity for binding some NR HBDs, it apparently does not influence the specificity of NR box preferences since the preference of GR HBD for NR box III over NR box II is evident even with GRIP1 NID fragments and small NR box peptides that lack NIDaux (29,31).
The GRIP1-(1011-1121) region has dual functions as an auxiliary HBD-binding region and as the primary CBP/p300binding domain. The minimum CBP/p300-binding region determined here is consistent with those reported previously for other members of the p160 coactivator family (9,20,33). Although we have not yet identified the specific structural features in this region that are responsible for the NIDaux function, we were able to rule out two obvious possibilities. Two leucine-rich motifs, previously named LCD4 and LCD5 (13), that partially resemble LXXLL motifs were not important for binding GR HBD. LCD4 is only partially conserved among the three different p160 family members, whereas LCD5 is more highly conserved. The LCD4 mutation also had no effect on GRIP1 binding to p300, but the LCD5 mutation substantially reduced p300 binding. Voegel et al. (33) found that a similar mutation in TIF2, the human orthologue to GRIP1, also reduced CBP/p300 binding. Since the LCD5 mutation reduced p300 binding without any effect on GR HBD binding, these two functions are genetically separable, i.e. the NIDaux function does not depend on p300 binding.