The PDZ domain of TIP-2/GIPC interacts with the C-terminus of the integrin alpha5 and alpha6 subunits.

Different cDNA libraries were screened by the yeast two-hybrid system using as a bait the cytoplasmic sequence of integrin alpha6A or alpha6B subunits. Surprisingly, the same PDZ domain-containing protein, TIP-2/GIPC, was isolated with either of the variants, although their sequences are different. Direct interaction assays with the cytoplasmic domain of the integrin alpha1--7 subunits revealed that in addition to alpha6A and alpha6B, TIP-2/GIPC reacted also with alpha5, but not other alpha integrin subunits. The specificity of the interaction was confirmed by in vitro protein binding assays with purified peptides corresponding to integrin cytoplasmic domains. Further analysis with either truncation fragments of TIP-2/GIPC or mutated integrin cytoplasmic domains indicated that the interaction occurs between the PDZ domain of TIP-2/GIPC and a consensus PDZ domain-binding sequence, SDA, present at the C-terminus of the integrin alpha5 and alpha6A subunits. The integrin alpha6B subunit terminates with a different sequence, SYS, which may represent a new PDZ domain-binding motif.     

5T4 Interacts with TIP-2/GIPC, a PDZ Protein, with Implications for Metastasis

Overexpression of the 5T4 transmembrane glycoprotein can have marked effects on both the actin cytoskeleton and cell migration. Using a yeast two-hybrid approach, we describe a novel interaction between 5T4 and TIP-2/GIPC, a cytoplasmic interacting protein containing a PDZ domain. The cytoplasmic tail of 5T4 contains a class I PDZ-binding motif (Ser-Asp-Val) and we demonstrate that this region, in particular the terminal valine, is required for 5T4 interaction with TIP-2/GIPC. HeLa cells expressing hemagglutinin-tagged TIP-2/GIPC (HA-TIP-2/GIPC) have an altered distribution of endogenous 5T4, which colocalizes with HA-TIP-2/GIPC, thus supporting an interaction. Furthermore, TIP-2/GIPC can be coimmunoprecipitated with 5T4 from HeLa cell lysates. Identification of the 5T4 and TIP-2/GIPC interaction provides the first link between 5T4 and the actin cytoskeleton. Since other proteins, like 5T4, associate with TIP-2/GIPC and are linked with cancer, we explore the possibility that TIP-2/GIPC may be a common factor involved in the cancer process.     

Human papillomavirus type 18 E6 protein binds the cellular PDZ protein TIP-2/GIPC, which is involved in transforming growth factor beta signaling and triggers its degradation by the proteasome

Several viral proteins expressed by DNA or RNA transforming viruses have the particular property of binding via their C-terminal end to various cellular proteins with PDZ domains. This study is focused on the PDZ protein TIP-2/GIPC, which was originally identified in two-hybrid screens performed with two different baits: the human T-cell leukemia virus type 1 Tax oncoprotein and the regulator of G signaling RGS-GAIP. Further studies have shown that TIP-2/GIPC is also able to associate with the cytoplasmic domains of various transmembrane proteins. In this report we show that TIP-2/GIPC interacts with the E6 protein of human papillomavirus type 18 (HPV-18). This event triggers polyubiquitination and proteasome-mediated degradation of the cellular protein. In agreement with this observation, silencing of E6 by RNA interference in HeLa cells causes an increase in the intracellular TIP-2/GIPC level. This PDZ protein has been previously found to be involved in transforming growth factor beta (TGF-beta) signaling by favoring expression of the TGF-beta type III receptor at the cell membrane. In line with this activity of TIP-2/GIPC, we observed that depletion of this protein in HeLa cells hampers induction of the Id3 gene by TGF-beta treatment and also diminishes the antiproliferative effect of this cytokine. Conversely, silencing of E6 increases the expression of Id3 and blocks proliferation of HeLa cells. These results support the notion that HPV-18 E6 renders cells less sensitive to the cytostatic effect of TGF-beta by lowering the intracellular amount of TIP-2/GIPC.     

Ancient ubiquitous protein 1 binds to the conserved membrane-proximal sequence of the cytoplasmic tail of the integrin alpha subunits that plays a crucial role in the inside-out signaling of alpha IIbbeta 3

Modification of the cytoplasmic tails of the integrin alpha(IIb)beta(3) plays an important role in the signal transduction in platelets. We searched for proteins that bind to the alpha(IIb) cytoplasmic tail using the yeast two-hybrid assay with a cDNA library of the megakaryocyte-derived cell line and identified a protein, ancient ubiquitous protein 1 (Aup1), that is ubiquitously expressed in human cells. Observation of UT7/TPO cells expressing a red fluorescent protein-tagged Aup1 indicated its localization in the cytoplasm. Immunoprecipitation of UT7/TPO cells by an antibody for Aup1 revealed that approximately 40% of alpha(IIb) is complexed with Aup1. Binding study with an alpha(IIb) cytoplasmic tail peptide and glutathione S-transferase-Aup1 fusion protein revealed a low affinity (K(d) = 90 microm). Subsequent yeast two-hybrid assay indicated binding of Aup1 to cytoplasmic tails of other integrin alpha subunits. Binding study with the purified Aup1 and various glutathione S-transferase-alpha(IIb) cytoplasmic tail peptides revealed specific binding of Aup1 to the membrane-proximal sequence (KVGFFKR) that is conserved among the integrin alpha subunits and plays a crucial role in the alpha(IIb)beta(3) inside-out signaling. As Aup1 possesses domains related to signal transduction, these results suggest involvement of Aup1 in the integrin signaling.     

Beta 3-endonexin, a novel polypeptide that interacts specifically with the cytoplasmic tail of the integrin beta 3 subunit

The adhesive and signaling functions of integrins are regulated through their cytoplasmic domains. We identified a novel 111 residue polypeptide, designated beta 3-endonexin, that interacted with the cytoplasmic tail of the beta 3 integrin subunit in a yeast two-hybrid system. This interaction is structurally specific, since it was reduced by 64% by a point mutation in the beta 3 cytoplasmic tail (S752-->P) that disrupts integrin signaling. Moreover, this interaction is integrin subunit specific since it was not observed with the cytoplasmic tails of the alpha IIb, beta 1, or beta 2 subunits. beta 3- Endonexin fusion proteins bound selectively to detergent-solubilized beta 3 from platelets and human umbilical vein endothelial cells, and beta 3-endonexin mRNA and protein were detected in platelets and other tissues. A related mRNA encoded a larger polypeptide that failed to bind to beta integrin tails. The apparent specificity of beta 3- endonexin for the beta 3 integrin subunit suggests potential mechanisms for selective modulation of integrin functions.     

In vitro interaction of a polypeptide homologous to human Ro/SS-A antigen (calreticulin) with a highly conserved amino acid sequence in the cytoplasmic domain of integrin alpha subunits

We endeavored to identify proteins interacting with KLGFFKR, a highly conserved motif in the cytoplasmic domain adjacent to the transmembrane domain of the alpha subunit of integrins. We found that affinity chromatography of cell extracts with this peptide followed by elution with EDTA resulted in the isolation of a 60-kDa protein (p60). The N-terminal amino acid sequence of this 60-kDa polypeptide was found to be highly homologous to the Ro/SS-A antigen, a 60-kDa protein homologous to calreticulin and Aplysia "memory molecule". The binding of p60 was found to be specific for the KLGFFKR sequence since this polypeptide did not bind to a peptide with a scrambled amino acid sequence (KLRFGFK), and it was also specifically eluted from the KLGFFKR affinity matrix ith soluble KLGFFKR peptide but not with the scrambled peptide. Solid phase in vitro binding assays demonstrated specific interaction of p60 with integrin alpha 3 and alpha 5 subunits but not with the beta 1 subunit. Furthermore, p60 could be copurified with alpha 3 beta 1 following coincubation in vitro. These interactions could be inhibited by KLGFFKR peptide and also by EDTA, indicating sequence-specific and divalent cation dependent binding. Despite the fact that calreticulin is thought to be localized in the endoplasmic reticulum, a pool of Ro/SS A antigen homologous 60-kDa polypeptide was found to be present in the soluble cytoplasm, indicating the feasibility of an interaction of p60 with the integrin alpha subunits. Our data suggest that p60 (Ro/SS-A Ag) can specifically bind to integrin alpha subunits via the highly conserved KLGFFKR amino acid sequence.     

GIPC interacts with the beta1-adrenergic receptor and regulates beta1-adrenergic receptor-mediated ERK activation

Beta1-adrenergic receptors, expressed at high levels in the human heart, have a carboxyl-terminal ESKV motif that can directly interact with PDZ domain-containing proteins. Using the beta1-adrenergic receptor carboxyl terminus as bait, we identified the novel beta1-adrenergic receptor-binding partner GIPC in a yeast two-hybrid screen of a human heart cDNA library. Here we demonstrate that the PDZ domain-containing protein, GIPC, co-immunoprecipitates with the beta1-adrenergic receptor in COS-7 cells. Essential for this interaction is the Ser residue of the beta1-adrenergic receptor carboxyl-terminal ESKV motif. Our data also demonstrate that beta1-adrenergic receptor stimulation activates the mitogen-activated protein kinase, ERK1/2. beta1-adrenergic receptor-mediated ERK1/2 activation was inhibited by pertussis toxin, implicating Gi, and was substantially decreased by the expression of GIPC. Expression of GIPC had no observable effect on beta1-adrenergic receptor sequestration or receptor-mediated cAMP accumulation. This GIPC effect was specific for the beta1-adrenergic receptor and was dependent on an intact PDZ binding motif. These data suggest that GIPC can regulate beta1-adrenergic receptor-stimulated, Gi-mediated, ERK activation while having no effect on receptor internalization or Gs-mediated cAMP signaling.     

The LIM-only protein DRAL/FHL2 binds to the cytoplasmic domain of several alpha and beta integrin chains and is recruited to adhesion complexes

LIM proteins contain one or more double zinc finger structures (LIM domains) mediating specific contacts between proteins that participate in the formation of multiprotein complexes. We report that the LIM-only protein DRAL/FHL2, with four and a half LIM domains, can associate with alpha(3A), alpha(3B), alpha(7A), and several beta integrin subunits as shown in yeast two-hybrid assays as well as after overexpression in human cells. The amino acid sequence immediately following the conserved membrane-proximal region in the integrin alpha subunits or the C-terminal region with the conserved NXXY motif of the integrin beta subunits are critical for binding DRAL/FHL2. Furthermore, the DRAL/FHL2 associates with itself and with other molecules that bind to the cytoplasmic domain of integrin alpha subunits. Deletion analysis of DRAL/FHL2 revealed that particular LIM domains or LIM domain combinations bind the different proteins. These results, together with the fact that full-length DRAL/FHL2 is found in cell adhesion complexes, suggest that it is an adaptor/docking protein involved in integrin signaling pathways.     

Identification of the alpha6 integrin as a candidate receptor for papillomaviruses.

Papillomaviruses (PVs) bind in a specific and saturable fashion to a range of epithelial and other cell lines. Treatment of cells with trypsin markedly reduces their ability to bind virus particles, suggesting that binding is mediated via a cell membrane protein. We have investigated the interaction of human PV type 6b L1 virus-like particles (VLPs) with two epithelial cell lines, CV-1 and HaCaT, which bind VLPs, and a B-cell line (DG75) previously shown not to bind VLPs. Immunoprecipitation of a mixture of PV VLPs with [35S]methionine-labeled cell extracts and with biotin-labeled cell surface proteins identified four proteins from CV-1 and HaCaT cells of 220, 120, 87, and 35 kDa that reacted with VLPs and were not present in DG75 cells. The alpha6beta4 integrin complex has subunits corresponding to the VLP precipitated proteins, and the tissue distribution of this complex suggested that it was a candidate human PV receptor. Monoclonal antibodies (MAbs) to the alpha6 or beta4 integrin subunits precipitated VLPs from a mixture of CV-1 cell proteins and VLPs, whereas MAbs to other integrin subunits did not. An alpha6 integrin-specific MAb (GoH3) inhibited VLP binding to CV-1 and HaCaT cells, whereas an anti-beta4 integrin MAb and a range of integrin-specific and other MAbs did not. Furthermore, human laminin, the natural ligand for the alpha6beta4 integrin, was able to block VLP binding. By use of sections of monkey esophagus, the distribution of alpha6 integrin expression in the basal epithelium was shown to coincide with the distribution of bound VLPs. Taken together, these data suggest that VLPs bind specifically to the alpha6 integrin subunit and that integrin complexes containing alpha6 integrin complexed with either beta1 or beta4 integrins may act as a receptor for PV binding and entry into epithelial cells.     

Identification of functional PDZ domain binding sites in several human proteins

TIP-15 was previously identified as a cellular protein that can bind to the C-terminal end of the HTLV-1 Tax protein via its two PDZ domains. The sequence of the N-terminal part of TIP-15 is identical to that of the synaptic protein PSD-95. Both proteins are likely to be produced from the same gene by alternative splicing. Whereas expression of the PSD-95 mRNA was detected only with brain RNAs, that of TIP-15 was detected with RNAs from thymus, brain, skeletal muscle and Jurkat cells. The TIP-15 protein exhibits an apparent molecular weight of 40 kD and is weakly expressed in T cell lines. A two-hybrid screen performed with TIP-15 as bait revealed the presence of a PDZ binding site (PDZ-BS) in the following proteins: Lysyl tRNA synthetase, 6-phosphogluconolactonase (6-GPL), Stress-activated protein kinase 3 (SAPK3), NET-1, Diacylglycerol kinase zeta, MTMR1, MCM7, and hSec8. The sequence at the C-terminal ends of these proteins matches the X-S/T-X-V-COOH consensus previously defined for PDZ-BSs, with the exception of 6-GPL and SAPK3 which include a leucine as the C-terminal residue. For Lysyl tRNA synthetase, NET1, MTMR1 and hSec8, binding to TIP-15 was confirmed by co-immunoprecipitation experiments performed with the extracts of transfected COS7 cells. These results show the existence of functional PDZ-BSs in these proteins, but future studies will be necessary to establish whether or not TIP-15 represents a physiological partner. The significance of the presence of a PDZ-BS in these various proteins is discussed with respect to their function.     

Molecular cloning of the rat integrin alpha 1-subunit: a receptor for laminin and collagen

Integrin heterodimers mediate a variety of adhesive interactions, including neuronal attachment to and process outgrowth on laminin. We report here the cloning and primary sequence of an M-200 kD integrin alpha subunit that associates with the integrin beta 1 subunit to form a receptor for both laminin and collagen. Similarities in ligand-binding specificity, relative molecular mass and NH2-terminal sequence make this a strong candidate for the rat homologue of the alpha subunit of the human integrin VLA-1. The full-length rat alpha 1 cDNAs encode a protein containing a purative signal sequence and a mature polypeptide of 1,152 amino acids, with extracellular, transmembrane and cytoplasmic domains. Several structural features are conserved with other integrin alpha chains, including (a) a sequence motif repeated seven times in the NH2-terminal half; (b) potential Ca2+/Mg2+ binding sites in repeats 5, 6, and 7, and (c) alignment of at least 14 of 23 cysteine residues. This rat alpha 1 sequence also contains a 206-amino acid I domain, inserted between repeats 2 and 3, that is homologous to I domains found in the same position in the alpha subunits of several integrins (VLA-2, Mac-1, LFA-1, p150). The rat alpha 1 and human VLA-2 apha subunits share greater than 50% sequence identity in the seven repeats and I domain, suggesting that these sequence identities may underlie some of their similar ligand-binding specificities. However, the rat integrin alpha 1 subunit has several unique features, including a 38-residue insert between two Ca2+/Mg2+ binding domains, and a divergent 15-residue cytoplasmic sequence, that may potentially account for unique functions of this integrin.     

Primary structure of the leukocyte function-associated molecule-1 alpha subunit: an integrin with an embedded domain defining a protein superfamily

The leukocyte function-associated molecule 1 (LFA-1, CD11a/CD18) is a membrane glycoprotein which functions in cell-cell adhesion by heterophilic interaction with intercellular adhesion molecule 1 (ICAM-1). LFA-1 consists of an alpha subunit (Mr = 180,000) and a beta subunit (Mr = 95,000). We report the molecular biology and protein sequence of the alpha subunit. Overlapping cDNAs containing 5,139 nucleotides were isolated using an oligonucleotide specified by tryptic peptide sequence. The mRNA of 5.5 kb is expressed in lymphoid and myeloid cells but not in a bladder carcinoma cell line. The protein has a 1,063-amino acid extracellular domain, a 29-amino acid transmembrane region, and a 53-amino acid cytoplasmic tail. The extracellular domain contains seven repeats. Repeats V-VII are in tandem and contain putative divalent cation binding sites. LFA-1 has significant homology to the members of the integrin superfamily, having 36% identity with the Mac-1 and p150,95 alpha subunits and 28% identity with other integrin alpha subunits. An insertion of approximately 200 amino acids is present in the NH2-terminal region of LFA-1. This "inserted/interactive" or I domain is also present in the p150,95 and Mac-1 alpha subunits but is absent from other integrin alpha subunits sequenced to date. The I domain has striking homology to three repeats in human von Willebrand factor, two repeats in chicken cartilage matrix protein, and a region of complement factor B. These structural features indicate a bipartite evolution from the integrin family and from an I domain family. These features may also correspond to relevant functional domains.     

Assembly of the stator in Escherichia coli ATP synthase. Complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha

Alpha subunit of Escherichia coli ATP synthase was expressed with a C-terminal 6-His tag and purified. Pure alpha was monomeric, was competent in nucleotide binding, and had normal N-terminal sequence. In F1 subunit dissociation/reassociation experiments it supported full reconstitution of ATPase, and reassociated complexes were able to bind to F1-depleted membranes with restoration of ATP-driven proton pumping. Therefore interaction between the stator delta subunit and the N-terminal residue 1-22 region of alpha occurred normally when pure alpha was complexed with other F1 subunits. On the other hand, three different types of experiments showed that no interaction occurred between pure delta and isolated alpha subunit. Unlike in F1, the N-terminal region of isolated alpha was not susceptible to trypsin cleavage. Therefore, during assembly of ATP synthase, complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha. We suggest that the N-terminal 1-22 residues of alpha are sequestered in isolated alpha until released by binding of beta to alpha subunit. This prevents 1/1 delta/alpha complexes from forming and provides a satisfactory explanation of the stoichiometry of one delta per three alpha seen in the F1 sector of ATP synthase, assuming that steric hindrance prevents binding of more than one delta to the alpha3/beta3 hexagon. The cytoplasmic fragment of the b subunit (bsol) did not bind to isolated alpha. It might also be that complexation of alpha with beta subunits is prerequisite for direct binding of stator b subunit to the F1-sector.     

Specific integrin subunits in bovine oocytes, including novel sequences for alpha 6 and beta 3 subunits

Integrins facilitate attachment of cells to the extra-cellular matrix, often binding the arginine-glycine-aspartic acid tri-peptide motif, thus facilitating cell migration, mediating cell-cell adhesion, linking the extracellular matrix (ECM) with cytoskeletal elements, and acting as signaling molecules. Adhesion activates signaling mechanisms that regulate integrin function, cytoskeletal assembly, cell behavior, and protein synthesis. Immunofluorescence was used to determine the presence of integrin alpha and beta subunits on the surface of bovine oocytes using a panel of monoclonal antibodies (mAbs) specific for alphaL, alphaM, alphaX, alphaV, alpha2, alpha4, alpha6, beta1, beta2, and beta3 antigens, with multiple antibodies for each subunit. Confocal microscopy indicated the presence of alphaV, alpha6, alpha4, alpha2, ss1, and ss3 integrin subunits on the plasma membrane of bovine oocytes. The presence of these subunits was verified by RT-PCR analysis using primers designed based on known gene sequences of bovine integrin subunits, or by using sequence information using bovine expressed sequence tags (EST) compared with known human and murine integrin subunit gene sequence information. Previously unpublished sequence information for bovine alpha6 and beta3 integrins was determined. The presence of these integrin subunits on the bovine oocyte vitelline membrane supports the hypothesis that sperm-oocyte interactions in the bovine are mediated by integrins.     

Regulation of alpha 6 beta 1 integrin laminin receptor function by the cytoplasmic domain of the alpha 6 subunit

The alpha 6 beta 1 integrin is expressed on the macrophage surface in an inactive state and requires cellular activation with PMA or cytokines to function as a laminin receptor (Shaw, L. M., J. M. Messier, and A. M. Mercurio. 1990. J. Cell Biol. 110:2167-2174). In the present study, the role of the alpha 6 subunit cytoplasmic domain in alpha 6 beta 1 integrin activation was examined. The use of P388D1 cells, an alpha 6-integrin deficient macrophage cell line, facilitated this analysis because expression of either the alpha 6A or alpha 6B subunit cDNAs restores their activation responsive laminin adhesion (Shaw, L. S., M. Lotz, and A. M. Mercurio. 1993. J. Biol. Chem. 268:11401-11408). A truncated alpha 6 cDNA, alpha 6-delta CYT, was constructed in which the human cytoplasmic domain sequence was deleted after the GFFKR pentapeptide. Expression of this cDNA in P388D1 cells resulted in the surface expression of a chimeric alpha 6-delta CYT beta 1 integrin that was unable to mediate laminin adhesion or increase this adhesion in response to PMA under normal conditions, i.e., in medium that contained physiological concentrations of Ca++ and Mg++. The alpha 6A-delta CYT transfectants adhered to laminin, however, when Ca++/Mg++ was replaced with 150 microM Mn++. We also assessed the role of serine phosphorylation in the regulation of alpha 6A beta 1 integrin function by site-directed mutagenesis of the two serine residues present in the alpha 6A cytoplasmic domain because this domain is phosphorylated on serine residues in response to stimuli that activate the laminin receptor function of alpha 6 A beta 1. Point mutations were introduced in the alpha 6A cDNA that changed either serine residue #1064 (M1) or serine residue #1071 (M2) to alanine residues. In addition, a double mutant (M3) was constructed in which both serine residues were changed to alanine residues. P388D1 transfectants which expressed these serine mutations adhered to laminin in response to PMA to the same extent as cells transfected with wild-type alpha 6A cDNA. These findings provide evidence for a novel mode of integrin regulation that is distinct from that reported for other regulated integrins (O'Toole, T. E., D. Mandelman, J. Forsyth, S. J. Shattil, E. F. Plow, and M. H. Ginsberg. 1991. Science (Wash. DC). 254:845-847. Hibbs, M. L., H. Xu, S. A. Stacker, and T. A. Springer. 1991. Science (Wash. DC). 251:1611-1613), and they demonstrate that serine phosphorylation of the alpha 6A cytoplasmic domain is not involved in this regulation.     

Binding of Paxillin to the alpha 9 Integrin Cytoplasmic Domain Inhibits Cell Spreading

alpha(9)beta(1) integrin is a member of the beta(1) integrin family, plays an important role in extravasation of neutrophils at sites of acute inflammation, and is required for the normal development of the lymphatic system. The alpha(9) and alpha(4) integrin subunits are most closely related and form a subfamily of integrin alpha subunits. Previously, we have reported that the alpha(4) cytoplasmic domain directly and tightly binds paxillin, an intracellular signaling adaptor molecule. This interaction accounts for some of the unusual functional responses to alpha(4) integrin-mediated cell adhesion, including stimulation of cell migration and inhibition of cell spreading and focal adhesion formation. In the current studies, we have examined the interaction between the alpha(9) cytoplasmic domain and paxillin. Here we report that the alpha(9) cytoplasmic domain binds paxillin directly and tightly and that the alpha(9)-paxillin association inhibits cell spreading. We have identified amino acid residues in the alpha(9) cytoplasmic domain, Trp(999) and Trp(1001), that are critical for paxillin binding, and alanine substitution of either Trp(999) or Trp(1001) blocks paxillin binding. Furthermore, these mutations also reverse the effect of the alpha(9) cytoplasmic domain on cell spreading. Thus, the alpha(9) and alpha(4) integrin subunits form a paxillin-binding subfamily of integrin alpha subunits, and direct binding of paxillin to the alpha(9) cytoplasmic domain mediates some of the biological activities of the alpha(9)beta(1) integrin.     

Identification of a residue in the gamma-aminobutyric acid type A receptor alpha subunit that differentially affects diazepam-sensitive and -insensitive benzodiazepine site binding

GABAA receptors that contain either the alpha4- or alpha6-subunit isoform do not recognize classical 1,4-benzodiazepines (BZDs). However, other classes of BZD site ligands, including beta-carbolines, bind to these diazepam-insensitive receptor subtypes. Some beta-carbolines [e.g. ethyl beta-carboline-3-carboxylate (beta-CCE) and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM)] display a higher affinity for alpha4- compared to alpha6-containing receptors. In order to identify the structural determinants that underlie these affinity differences, we constructed chimeric alpha6/alpha4 subunits and co-expressed these with wild-type rat beta2 and gamma2L subunits in tsA201 cells for radioligand binding analysis. After identification of candidate regions, site-directed mutagenesis was used to narrow the ligand selectivity to a single amino acid residue (alpha6N204/alpha4I203). Substitutions at alpha6N204 did not alter the affinity of the imidazobenzodiazepine Ro15-4513. A homologous mutation in the diazepam-sensitive alpha1 subunit (S205N) resulted in a 7-8-fold reduction in affinity for the beta-carbolines examined. Although the binding of the classical agonist flunitrazepam was relatively unaffected by this mutation in the alpha1 subunit, the affinity for Ro15-1788 and Ro15-4513 was decreased by approximately 19-fold and approximately 38-fold respectively. The importance of this residue, located in the Loop C region of the extracellular N-terminus of the subunit protein, emphasizes the differential interaction of ligands with the alpha subunit in diazepam-sensitive and -insensitive receptors.     

Syntrophins regulate alpha1D-adrenergic receptors through a PDZ domain-mediated interaction

To find novel cytoplasmic binding partners of the alpha1D-adrenergic receptor (AR), a yeast two-hybrid screen using the alpha1D-AR C terminus as bait was performed on a human brain cDNA library. Alpha-syntrophin, a protein containing one PDZ domain and two pleckstrin homology domains, was isolated in this screen as an alpha1D-AR-interacting protein. Alpha-syntrophin specifically recognized the C terminus of alpha1D- but not alpha1A- or alpha1B-ARs. In blot overlay assays, the PDZ domains of syntrophin isoforms alpha, beta1, and beta2 but not gamma1 or gamma2 showed strong selective interactions with the alpha1D-AR C-tail fusion protein. In transfected human embryonic kidney 293 cells, full-length alpha1D- but not alpha1A- or alpha1B-ARs co-immunoprecipitated with syntrophins, and the importance of the receptor C terminus for the alpha1D-AR/syntrophin interaction was confirmed using chimeric receptors. Mutation of the PDZ-interacting motif at the alpha1D-AR C terminus markedly decreased inositol phosphate formation stimulated by norepinephrine but not carbachol in transfected HEK293 cells. This mutation also dramatically decreased alpha1D-AR binding and protein expression. In addition, stable overexpression of alpha-syntrophin significantly increased alpha1D-AR protein expression and binding but did not affect those with a mutated PDZ-interacting motif, suggesting that syntrophin plays an important role in maintaining receptor stability by directly interacting with the receptor PDZ-interacting motif. This direct interaction may provide new information about the regulation of alpha1D-AR signaling and the role of syntrophins in modulating G protein-coupled receptor function.