A transmembrane leucine zipper is required for activation of the dimeric receptor tyrosine kinase DDR1

Receptor tyrosine kinases of the discoidin domain family, DDR1 and DDR2, are activated by different types of collagen and play important roles in cell adhesion, migration, proliferation, and matrix remodeling. In a previous study, we found that collagen binding by the discoidin domain receptors (DDRs) requires dimerization of their extracellular domains (Leitinger, B. (2003) J. Biol. Chem. 278, 16761-16769), indicating that the paradigm of ligand-induced receptor dimerization may not apply to the DDRs. Using chemical cross-linking and co-immunoprecipitation of differently tagged DDRs, we now show that the DDRs form ligand-independent dimers in the biosynthetic pathway and on the cell surface. We further show that both the extracellular and the cytoplasmic domains are individually dispensable for DDR1 dimerization. The DDR1 transmembrane domain contains two putative dimerization motifs, a leucine zipper and a GXXXG motif. Mutations disrupting the leucine zipper strongly impaired collagen-induced transmembrane signaling, although the mutant DDR1 proteins were still able to dimerize, whereas mutation of the GXXXG motif had no effect. A bacterial reporter assay (named TOXCAT) showed that the DDR1 transmembrane domain has a strong potential for self-association in a biological membrane and that this interaction occurs via the leucine zipper and not the GXXXG motif. Our results demonstrate that the DDRs exist as stable dimers in the absence of ligand and that receptor activation requires specific interactions made by the transmembrane leucine zipper.     

Mutational studies of G553 in TM5 of ABCG2: a residue potentially involved in dimerization

ABCG2 is an ATP-binding cassette half-transporter conferring resistance to chemotherapeutic agents such as mitoxantrone, irinotecan, and flavopiridol. With its one transmembrane and one ATP-binding domain, ABCG2 is thought to homodimerize for function. One conserved region potentially involved in dimerization is a three-amino acid sequence in transmembrane segment 5 (residues 552-554). Mutations in the corresponding residues in the Drosophila white protein (an orthologue of ABCG2) are thought to disrupt heterodimerization. We substituted glycine 553 with leucine (G553L) followed by stable transfection in HEK 293 cells. The mutant was not detectable on the cell surface, and markedly reduced protein expression levels were observed by immunoblotting. A deficiency in N-linked glycosylation was suggested by a reduction in molecular mass compared to that of the 72 kDa wild-type ABCG2. Similar results were observed with the G553E mutant. Confocal microscopy demonstrated mostly ER localization of the G553L mutant in HEK 293 cells, even when coexpressed with the wild-type protein. Despite its altered localization, the G553L and G553E mutants were cross-linked using amine-reactive cross-linkers with multiple arm lengths, suggesting that the monomers are in the proximity of each other but are unable to complete normal trafficking. Interestingly, when expressed in Sf9 insect cells, G553L moves to the cell membrane but is unable to hydrolyze ATP or transport the Hoechst dye. Still, when coexpressed, the mutant interferes with the Hoechst transport activity of the wild-type protein. These data show that glycine 553 is important for protein trafficking and are consistent with, but do not yet prove, its involvement in ABCG2 homodimerization.     

Mutational analysis of the role of GXXXG motif in the function of human organic anion transporter 1 (hOAT1)

Human organic anion transporter hOAT1 plays a critical role in the body disposition of environmental toxins and clinically important drugs including anti-HIV therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories. hOAT1 has two GXXXG motifs in its transmembrane domains 2 and 5, a motif linked to the protein processing and oligomerization of other proteins. In the current study, we substituted glycine of these GXXXG motifs with alanine and evaluated the effect of such mutations on the expression and function of hOAT1. Mutations of GXXXG motif in the transmembrane domain 2 resulted in mutants G144A and G148A, both of which had no transport activity due to complete loss in the surface and total cell expression of the transporter protein. Treatment of G144A- and G148A-expressing cells with proteasomal inhibitor resulted in the recovery of ER-resident immature form of hOAT1, but not its surface-resident mature form, whereas treatment of these cells with lysosomal inhibitor had no effect on the expression of the mutant transporters. Mutations of GXXXG motif in the transmembrane domain 5 resulted in mutants G223A and G227A, among which only G227 had dramatic reduction of transport activity due to dramatic loss in the surface and total cell expression of the transporter. The reduction in the surface expression of G227 was consistent with the decrease in maximum transport velocity Vmax. Treatment of G227A-expressing cells with proteasomal inhibitor or lysosomal inhibitor resulted in partial recovery of both the immature form and the mature form of hOAT1 in the total cell extracts. However, such partial recovery of the mature form in total cell extracts did not lead to the partial recovery of surface expression and function of the transporter. Our data suggest that the GXXXG motifs in transmembrane domains 2 and 5 play critical roles in the stability of hOAT1.     

Characterization of the GXXXG motif in the first transmembrane segment of Japanese encephalitis virus precursor membrane (prM) protein

The interaction between prM and E proteins in flavivirus-infected cells is a major driving force for the assembly of flavivirus particles. We used site-directed mutagenesis to study the potential role of the transmembrane domains of the prM proteins of Japanese encephalitis virus (JEV) in prM-E heterodimerization as well as subviral particle formation. Alanine insertion scanning mutagenesis within the GXXXG motif in the first transmembrane segment of JEV prM protein affected the prM-E heterodimerization; its specificity was confirmed by replacing the two glycines of the GXXXG motif with alanine, leucine and valine. The GXXXG motif was found to be conserved in the JEV serocomplex viruses but not other flavivirus groups. These mutants with alanine inserted in the two prM transmembrane segments all impaired subviral particle formation in cell cultures. The prM transmembrane domains of JEV may play importation roles in prM-E heterodimerization and viral particle assembly.     

Oligomerization, biogenesis, and signaling is promoted by a glycophorin A-like dimerization motif in transmembrane domain 1 of a yeast G protein-coupled receptor

G protein-coupled receptors (GPCRs) can form dimeric or oligomeric complexes in vivo. However, the functions and mechanisms of oligomerization remain poorly understood for most GPCRs, including the alpha-factor receptor (STE2 gene product) of the yeast Saccharomyces cerevisiae. Here we provide evidence indicating that alpha-factor receptor oligomerization involves a GXXXG motif in the first transmembrane domain (TM1), similar to the transmembrane dimerization domain of glycophorin A. Results of fluorescence resonance energy transfer, fluorescence microscopy, endocytosis assays of receptor oligomerization in living cells, and agonist binding assays indicated that amino acid substitutions affecting the glycine residues of the GXXXG motif impaired alpha-factor receptor oligomerization and biogenesis in vivo but did not significantly impair agonist binding affinity. Mutant receptors exhibited signaling defects that were not due to impaired cell surface expression, indicating that oligomerization promotes alpha-factor receptor signal transduction. Structure-function studies suggested that the GXXXG motif in TM1 of the alpha-factor receptor promotes oligomerization by a mechanism similar to that used by the GXXXG dimerization motif of glycophorin A. In many mammalian GPCRs, motifs related to the GXXXG sequence are present in TM1 or other TM domains, suggesting that similar mechanisms are used by many GPCRs to form dimers or oligomeric arrays.     

Characterization of drug transport,ATP hydrolysis,and nucleotide trapping by the human ABCG2 multidrug transporter. Modulation of substrate specificity by a point mutation

The overexpression of the human ATP-binding cassette half-transporter, ABCG2 (placenta-specific ABC transporter, mitoxantrone resistance-associated protein, breast cancer resistance protein), causes multidrug resistance in tumor cells. An altered drug resistance profile and substrate recognition were suggested for wild-type ABCG2 and its mutant variants (R482G and R482T); the mutations were found in drug-selected tumor cells. In order to characterize the different human ABCG2 transporters without possible endogenous dimerization partners, we expressed these proteins and a catalytic center mutant (K86M) in Sf9 insect cells. Transport activity was followed in intact cells, whereas the ATP binding and hydrolytic properties of ABCG2 were studied in isolated cell membranes. We found that the K86M mutant had no transport or ATP hydrolytic activity, although its ATP binding was retained. The wild-type ABCG2 and its variants, R482G and R482T, showed characteristically different drug and dye transport activities; mitoxantrone and Hoechst 33342 were transported by all transporters, whereas rhodamine 123 was only pumped by the R482G and R482T mutants. In each case, ABCG2-dependent transport was blocked by the specific inhibitor, fumitremorgin C. A relatively high basal ABCG2-ATPase, inhibited by fumitremorgin C, was observed in all active proteins, but specific drug stimulation could only be observed in the case of R482G and R482T mutants. We found that ABCG2 is capable of a vanadate-dependent adenine nucleotide trapping. Nucleotide trapping was stimulated by the transported compounds in the R482G and R482T variants but not in the wild-type ABCG2. These experiments document the applicability of the Sf9 expression system for parallel, quantitative examination of the specific transport and ATP hydrolytic properties of different ABCG2 proteins and demonstrate significant differences in their substrate interactions.     

The yeast F(1)F(0)-ATP synthase: analysis of the molecular organization of subunit g and the importance of a conserved GXXXG motif

The F(1)F(0)-ATP synthase enzyme is located in the inner mitochondrial membrane, where it forms dimeric complexes. Dimerization of the ATP synthase involves the physical association of the neighboring membrane-embedded F(0)-sectors. In yeast, the F(0)-sector subunits g and e (Su g and Su e, respectively) play a key role in supporting the formation of ATP synthase dimers. In this study we have focused on Su g to gain a better understanding of the function and the molecular organization of this subunit within the ATP synthase complex. Su g proteins contain a GXXXG motif (G is glycine, and X is any amino acid) in their single transmembrane segment. GXXXG can be a dimerization motif that supports helix-helix interactions between neighboring transmembrane segments. We demonstrate here that the GXXXG motif is important for the function and in particular for the stability of Su g within the ATP synthase. Using site-directed mutagenesis and cross-linking approaches, we demonstrate that Su g and Su e interact, and our findings emphasize the importance of the membrane anchor regions of these proteins for their interaction. Su e also contains a conserved GXXXG motif in its membrane anchor. However, data presented here would suggest that an intact GXXXG motif in Su g is not essential for the Su g-Su e interaction. We suggest that the GXXXG motif may not be the sole basis for a Su g-Su e interaction, and possibly these dimerization motifs may enable both Su g and Su e to interact with another mitochondrial protein.     

The transmembrane domain of CEACAM1-4S is a determinant of anchorage independent growth and tumorigenicity

CEACAM1 is a multifunctional Ig-like cell adhesion molecule expressed by epithelial cells in many organs. CEACAM1-4L and CEACAM1-4S, two isoforms produced by differential splicing, are predominant in rat liver. Previous work has shown that downregulation of both isoforms occurs in rat hepatocellular carcinomas. Here, we have isolated an anchorage dependent clone, designated 253T-NT that does not express detectable levels of CEACAM1. Stable transfection of 253-NT cells with a wild type CEACAM1-4S expression vector induced an anchorage independent growth in vitro and a tumorigenic phenotype in vivo. These phenotypes were used as quantifiable end points to examine the functionality of the CEACAM1-4S transmembrane domain. Examination of the CEACAM1 transmembrane domain showed N-terminal GXXXG dimerization sequences and C-terminal tyrosine residues shown in related studies to stabilize transmembrane domain helix-helix interactions. To examine the effects of transmembrane domain mutations, 253-NT cells were transfected with transmembrane domain mutants carrying glycine to leucine or tyrosine to valine substitutions. Results showed that mutation of transmembrane tyrosine residues greatly enhanced growth in vitro and in vivo. Mutation of transmembrane dimerization motifs, in contrast, significantly reduced anchorage independent growth and tumorigenicity. 253-NT cells expressing CEACAM1-4S with both glycine to leucine and tyrosine to valine mutations displayed the growth-enhanced phenotype of tyrosine mutants. The dramatic effect of transmembrane domain mutations constitutes strong evidence that the transmembrane domain is an important determinant of CEACAM1-4S functionality and most likely by other proteins with transmembrane domains containing dimerization sequences and/or C-terminal tyrosine residues.     

Structure and function analysis of CaMdr1p, a major facilitator superfamily antifungal efflux transporter protein of Candida albicans: identification of amino acid residues critical for drug/H+ transport

We have cloned and overexpressed multidrug transporter CaMdr1p as a green fluorescent protein-tagged protein to show its capability to extrude drug substrates. The drug extrusion was sensitive to pH and energy inhibitors and displayed selective substrate specificity. CaMdr1p has a unique and conserved antiporter motif, also called motif C [G(X6)G(X3)GP(X2)GP(X2)G], in its transmembrane segment 5 (TMS 5). Alanine scanning of all the amino acids of the TMS 5 by site-directed mutagenesis highlighted the importance of the motif, as well as that of other residues of TMS 5, in drug transport. The mutant variants of TMS 5 were placed in four different categories. The first category had four residues, G244, G251, G255, and G259, which are part of the conserved motif C, and their substitution with alanine resulted in increased sensitivity to drugs and displayed impaired efflux of drugs. Interestingly, first category mutants, when replaced with leucine, resulted in more dramatic loss of drug resistance and efflux. Notwithstanding the location in the core motif, the second category included residues which are part of the motif, such as P260, and those which were not part of the motif, such as L245, W248, P256, and F262, whose substitution with alanine resulted in a severe loss of drug resistance and efflux. The third category included G263, which is a part of motif C, but unlike other conserved glycines, its replacement with alanine or leucine showed no change in the phenotype. The replacement of the remaining 11 residues of the fourth category did not result in any change. The putative helical wheel projection showed clustering of functionally critical residues to one side and thus suggests an asymmetric nature of TMS 5.     

Mutations of the central tyrosines of putative cholesterol recognition amino acid consensus (CRAC) sequences modify folding,activity, and sterol-sensing of the human ABCG2 multidrug transporter

Human ABCG2 is a plasma membrane glycoprotein causing multidrug resistance in cancer. Membrane cholesterol and bile acids are efficient regulators of ABCG2 function, while the molecular nature of the sterol-sensing sites has not been elucidated. The cholesterol recognition amino acid consensus (CRAC, L/V-(X)_(1–5)-Y-(X)_(1–5)-R/K) sequence is one of the conserved motifs involved in cholesterol binding in several proteins. We have identified five potential CRAC motifs in the transmembrane domain of the human ABCG2 protein. In order to define their roles in sterol-sensing, the central tyrosines of these CRACs (Y413, 459, 469, 570 and 645) were mutated to S or F and the mutants were expressed both in insect and mammalian cells. We found that mutation in Y459 prevented protein expression; the Y469S and Y645S mutants lost their activity; while the Y570S, Y469F, and Y645F mutants retained function as well as cholesterol and bile acid sensitivity. We found that in the case of the Y413S mutant, drug transport was efficient, while modulation of the ATPase activity by cholesterol and bile acids was significantly altered. We suggest that the Y413 residue within a putative CRAC motif has a role in sterol-sensing and the ATPase/drug transport coupling in the ABCG2 multidrug transporter.     

Use of peptide antibodies to probe for the mitoxantrone resistance-associated protein MXR/BCRP/ABCP/ABCG2

Recent studies have characterized the ABC half-transporter associated with mitoxantrone resistance in human cancer cell lines. Encoded by the ABCG2 gene, overexpression confers resistance to camptothecins, as well as to mitoxantrone. We developed four polyclonal antibodies against peptides corresponding to four different epitopes on the mitoxantrone resistance-associated protein, ABCG2. Three epitopes localized on the cytoplasmic region of ABCG2 gave rise to high-affinity antibodies, which were demonstrated to be specific for ABCG2. Western blot analysis of cells with high levels of ABCG2 showed a single major band of the expected 72-kDa molecular size of ABCG2 under denaturing conditions. Immunoblot analysis performed under non-reducing conditions and after treatment with cross-linking reagents demonstrated a molecular weight shift from 72 kDa to several bands of 180 kDa and higher molecular weight, suggesting detection of dimerization products of ABCG2. Evidence of N-linked glycosylation was also obtained using tunicamycin and N-glycosidase F. Finally, both by light, fluorescence and electron microscopic immunohistochemical staining, we demonstrate cytoplasmic and predominantly plasma membrane localization of ABCG2 in cell lines with high levels of expression. Plasma membrane staining was observed on the surface of the chorionic villi in placenta. These results support the hypothesis that ABCG2 is an ABC half-transporter that forms dimers in the plasma membrane, functioning as an ATP-dependent outward pump for substrate transport.     

Sinorhizobium meliloti dctA mutants with partial ability to transport dicarboxylic acids

Sinorhizobium meliloti dctA encodes a transport protein needed for a successful nitrogen-fixing symbiosis between the bacteria and alfalfa. Using the toxicity of the DctA substrate fluoroorotic acid as a selective agent in an iterated selection procedure, four independent S. meliloti dctA mutants were isolated that retained some ability to transport dicarboxylates. Two mutations were located in a region called motif B located in a predicted transmembrane helix of the protein that has been shown in other members of the glutamate transporter family to be involved in cation binding. A G114D mutation was located in the third transmembrane helix, which had not previously been directly implicated in transport. Multiple sequence alignment of more than 60 members of the glutamate transporter family revealed a glycine at this position in nearly all members of the family. The fourth mutant was able to transport succinate at almost wild-type levels but was impaired in malate and fumarate transport. It contains two mutations: one in a periplasmic domain and the other predicted to be in the cytoplasm. Separation of the mutations showed that each contributed to the altered substrate preference. dctA deletion mutants that contain the mutant dctA alleles on a plasmid can proceed further in symbiotic development than null mutants of dctA, but none of the plasmids could support symbiotic nitrogen fixation, although they can transport dicarboxylates, some at relatively high levels.     

Mutations within an intramembrane leucine heptad repeat disrupt oligomer formation of the rat GABA transporter 1

Na(+)/Cl(-)-dependent neurotransmitter transporters form constitutive oligomers, the significance of which is not known. In soluble proteins, leucine heptad repeats drive dimerization; the rat gamma-aminobutyric acid transporter GAT-1 (rGAT) contains a motif reminiscent of a leucine heptad repeat in the second transmembrane helix (TM2). We substituted leucine residues in TM2 of rGAT by alanine and tested the ability of the resulting mutants to form oligomers by three methods of F?rster resonance energy transfer (FRET) microscopy. Replacement of one leucine (L97A) resulted in considerable loss of energy transfer, replacing two or more ablated it completely. Furthermore, intracellular trapping increased with the number of leucine substitutions. Only rGAT-L97A reached the cell surface to a sufficient amount such that, in intact cells, it was indistinguishable from wild type rGAT with respect to substrate transport, binding of inhibitors, and regulation by protein kinase C. However, in membrane vesicles prepared from transfected cells, all mutants were still functional. In addition, FRET was readily detected during maturation of wild type rGAT, when the bulk of the protein resided in the endoplasmic reticulum. Hence, our findings strongly argue for a role of oligomer formation during biosynthesis and subsequent delivery of the multimer from the endoplasmic reticulum to the plasma membrane.     

Identification of a functionally critical GXXG motif and its relationship to the folate binding site of the proton-coupled folate transporter (PCFT-SLC46A1)

The proton-coupled folate transporter (PCFT) mediates intestinal folate absorption, and loss-of-function mutations in this gene result in the autosomal recessive disorder hereditary folate malabsorption. The current study, focused on a structure-functional analysis of this transporter, identified Gly-189 and Gly-192 (a GxxG motif) located in the fifth transmembrane domain as residues that could not be replaced with alanine without a loss of function. In contrast, function was preserved when Gly-56 and Gly-59 (the other conservative GXXG motif in human PCFT) were replaced with alanine. Similarly, Gly-93 and Gly-97, which constitute the only conserved GXXXG dimerization motif in human PCFT, tolerated alanine substitution. To explore the role of this region in folate binding, the residues around Gly-189 and Gly-192 were analyzed by the substituted cysteine accessibility method. Both I188C and M193C mutants were functional and were inhibited by membrane-impermeable sulfhydryl-reactive reagents; this could be prevented with PCFT substrate, but the protection was sustained at 0°C only for the I188C mutant, consistent with localization of Ile-188 in the PCFT folate binding pocket. The functional role of residues around Gly-189 and Gly-192 is consistent with a molecular structural model in which these two residues along with Ieu-188 are accessible to the PCFT aqueous translocation pathway.     

Arabidopsis ABCG Transporters,Which Are Required for Export of Diverse Cuticular Lipids,Dimerize in Different Combinations

ATP binding cassette (ABC) transporters play diverse roles, including lipid transport, in all kingdoms. ABCG subfamily transporters that are encoded as half-transporters require dimerization to form a functional ABC transporter. Different dimer combinations that may transport diverse substrates have been predicted from mutant phenotypes. In Arabidopsis thaliana, mutant analyses have shown that ABCG11/WBC11 and ABCG12/CER5 are required for lipid export from the epidermis to the protective cuticle. The objective of this study was to determine whether ABCG11 and ABCG12 interact with themselves or each other using bimolecular fluorescence complementation (BiFC) and protein traffic assays in vivo. With BiFC, ABCG11/ABCG12 heterodimers and ABCG11 homodimers were detected, while ABCG12 homodimers were not. Fluorescently tagged ABCG11 or ABCG12 was localized in the stem epidermal cells of abcg11 abcg12 double mutants. ABCG11 could traffic to the plasma membrane in the absence of ABCG12, suggesting that ABCG11 is capable of forming flexible dimer partnerships. By contrast, ABCG12 was retained in the endoplasmic reticulum in the absence of ABCG11, indicating that ABCG12 is only capable of forming a dimer with ABCG11 in epidermal cells. Emerging themes in ABCG transporter biology are that some ABCG proteins are promiscuous, having multiple partnerships, while other ABCG transporters form obligate heterodimers for specialized functions.     

Effects of α-adrenoceptor antagonists on ABCG2/BCRP-mediated resistance and transport

Acquired resistance of cancer cells to various chemotherapeutic agents is known as multidrug resistance, and remains a critical factor in the success of cancer treatment. It is necessary to develop the inhibitors for multidrug resistance. The aim of this study was to examine the effects of eight α-adrenoceptor antagonists on ABCG2/BCRP-mediated resistance and transport. Previously established HeLa/SN100 cells, which overexpress ABCG2/BCRP but not ABCB1/MDR1, were used. The effects of the antagonists on sensitivity to mitoxantrone and the transport activity of Hoehst33342, both substrates for ABCG2/BCRP, were evaluated using the WST-1 assay and cellular kinetics, respectively. ABCG2/BCRP mRNA expression and the cell cycle were also examined by real-time RT-PCR and flow cytometry, respectively. Sensitivity to mitoxantrone was reversed by the α-adrenoceptor antagonists in a concentration-dependent manner, although such effects were also found in the parental HeLa cells. Levels of ABCG2/BCRP mRNA expression were not influenced by the antagonists. The transport activity of Hoechst33342 was decreased by doxazosin and prazosin, but unaffected by the other antagonists. In addition, doxazosin and prazosin increased the proportion of S phase cells in the cultures treated with mitoxantrone, whereas the other α-adrenoceptor antagonists increased the percentage of cells in G(2)/M phase. These findings suggested that doxazosin and prazosin reversed resistance mainly by inhibiting ABCG2/BCRP-mediated transport, but the others affected sensitivity to mitoxantrone via a different mechanism.     

Complex interactions at the helix-helix interface stabilize the glycophorin A transmembrane dimer

To explore the residue interactions in the glycophorin A dimerization motif, an alanine scan double mutant analysis at the helix-helix interface was carried out. These data reveal a combination of additive and coupled effects. The majority of the double mutants are found to be equally or slightly more stable than would be predicted by the sum of the energetic cost of the single-point mutants. The proximity of the mutated sites is not related to the presence of coupling between those sites. Previous studies reveal that a single face of the glycophorin A monomer contains a specific glycine-containing motif (GxxxG) that is thought to be a driving force for the association of transmembrane helices. Double mutant cycles suggest that the relationship of the GxxxG motif to the remainder of the helix-helix interface is complex. Sequences containing mutations that abolish the GxxxG motif retain an ability to dimerize, while a sequence containing a GxxxG motif appears unable to form dimers. The energetic effects of weakly coupled and additive double mutants can be explained by changes in van der Waals interactions at the dimer interface. These results emphasize the fact that the sequence context of the dimer interface modulates the strength of the glycophorin A GxxxG-mediated transmembrane dimerization reaction.