Androgen binding profiles of two distinct nuclear androgen receptors in Atlantic croaker (Micropogonias undulatus)

In the present study, the binding affinities of 28 androgens for two nuclear androgen receptors (AR), termed AR1 and AR2, in Atlantic croaker (Micropogonias undulatus) brain and ovarian tissues, respectively, were determined using competitive binding assays. The 5alpha-reduction of steroids, in general, increased the metabolite's binding affinity for AR2 while decreasing it for AR1. In addition, few androgens bound to AR1 with high affinity and modifications to the basic 3-ketone,4-ene,17beta-hydroxy structure of testosterone usually reduced its binding affinity for AR1. However, androgens with ketone groups at the 3- and 17-position bound with high affinity to AR1 provided that the androgen had either a 5alpha-reduced A-ring or a third ketone group at the 11-position. This suggests that there may be several high affinity conformations that AR1 can occupy depending upon whether an androgen possesses a ketone or a hydroxyl group at the 17-position. The binding of androgens to AR2 showed a more predictable pattern, 5alpha-reduced steroids bound better than 4-ene steroids and any changes to the basic 3-keto,17-hydroxy motif of 5alpha-dihydrotestosterone lowered the binding affinity of a steroid. However, these structural changes often caused only minor decreases in binding affinity, such that AR2 has a broader affinity for androgens and a greater affinity than AR1 for structurally diverse androgens. Widely different androgen binding affinities of AR1 and AR2 suggest that these two nuclear androgen receptors may mediate the physiological actions of different androgens in teleosts.     

Metabolism of trimethylamines in kelp bass (Paralabrax clathratus) and marine and freshwater pink salmon (Oncorhynchus gorbuscha)

Summary ³H or¹⁴C labeled tracers were used to investigate the metabolism of trimethylamine (TMA), trimethylamine oxide (TMAO), choline, and betaine in free swimming kelp bass (Paralabrax clathratus). An indwelling cannula in the ventral aorta was used to administer tracer and withdraw blood samples. The concentrations of TMA and TMAO were determined in liver, muscle, and plasma. The TMA liver content is higher than that of muscle (0.85 vs 0.01 moles/g wet tissue) while the amount of TMAO in muscle greatly exceeds its liver concentration (60 vs 0.04 moles/g wet tissue). Prolonged fasting (21 and 75 days) or feeding the fish a squid diet containing high levels of TMAO did not alter the tissue concentrations of TMA or TMAO, suggesting that these compounds are endogenous in origin and that their tissue concentrations are subject to regulation. Comparison of the radiospecific activities of TMA and TMAO, and the administered TMA tracer suggest that TMA is channled directly to TMAO in the liver without equilibration in the hepatic TMA pool. The conversion kinetics of TMA to TMAO and the distribution of these amines in liver and muscle with time suggest that labeled TMA is rapidly taken up into a sequestered pool from which it is slowly released, oxidized to TMAO in the liver, and then transported via the circulation to the muscle mass. The location of this proposed sequestered TMA pool was not determined. Experiments with labeled choline and betaine suggest that these compounds are interconverted in the liver and that enzymes are present for conversion of choline betaine TMA TMAO. Labeled dimethylamine (DMA) was not metabolized and is, therefore, probably not a precursor of TMA and TMAO. [¹⁴C]Trimethylamine (TMA) was also used to investigate the possible role of trimethylamine oxide (TMAO) as an osmoregulatory compound in migrating prespawning cannulated Pacific pink salmon (Oncorhynchus gorbuscha) taken from marine or fresh water environments. Marine and fresh water salmon oxidized administered [¹⁴C]TMA to TMAO; labeled metabolites other than TMA and TMAO were not detected. Four hours after [¹⁴C]TMA injection about 10% of the administered dose was present in muscle as labeled TMAO and about 33% as TMA. Unlike our finding in kelp bass, [¹⁴C]TMAO was not recovered in liver, although low amounts of labeled TMA were found (0.4% of administered dose). Labeled TMA and TMAO, however, were detected in liver after [¹⁴C]betaine adminstration to a marine salmon, indicating that TMA-mono-oxygenase is present in salmon liver. The presence of labeled choline indicates that choline and betaine are interconverted as in kelp bass. The amount of [¹⁴C]TMA oxidized to [¹⁴C]TMAO and then accumulated in the muscle mass is the same in marine and fresh water salmon, as is the amount of chemical TMAO present (4.6 moles/g muscle).     

Binding characteristics of estrogen receptor (ER) in Atlantic croaker (Micropogonias undulatus) testis: different affinity for estrogens and xenobiotics from that of hepatic ER.

An estrogen receptor (ER) was identified in cytosolic and nuclear fractions of the testis in a marine teleost, Atlantic croaker (Micropogonias undulatus). A single class of high affinity, low capacity, and displaceable binding sites was identified by saturation analysis, with a Kd of 0.40 nM in cytosolic extracts and a Kd of 0.33 nM in nuclear extracts. Competition studies demonstrated that the receptor was highly specific for estrogens (diethylstilbestrol > estradiol > estriol = estrone) and also bound several antiestrogens. Testosterone and 5alpha-dihydrotestosterone had much lower affinities for the receptor, whereas no displacement of specific binding occurred with 11-ketotestosterone or any of the C21 maturation-inducing steroids. A variety of xenoestrogens, including o,p'-dichlorodiphenyltrichloroethane (DDT), chlordecone (Kepone), nonylphenol, hydroxylated polychlorinated biphenyls (PCBs), and the mycotoxin zearalenone, bound to the receptor with relatively low binding affinities, 10(-3) to 10(-5) that of estradiol. A comparison of the binding affinities of various ligands for the testicular ER and the hepatic ER in this species revealed that the testicular ER was saturated at a lower [3H]estradiol concentration (1 nM vs. 4 nM). The binding affinities of several compounds, including testosterone and nafoxidine, exhibited marked differences for the two ERs; and most of the estrogens and xenoestrogens tested had higher binding affinities for the testicular receptor. Minor amounts of estradiol (0.12 ng/g tissue/h) were produced by testicular tissue fragments incubated in vitro, and estradiol was detected in male Atlantic croaker plasma. The identification of a testicular ER and evidence that estradiol is produced by the testes in croaker suggest that estrogens participate in the hormonal control of testicular function in teleosts.     

New species of Cardicola (Digenea: Aporocotylidae) from heart of Atlantic croaker, Micropogonias undulatus (Perciformes: Sciaenidae), of the South Atlantic Bight

Cardicola parvus n. sp. (Digenea: Aporocotylidae) infects the heart of Atlantic croaker, Micropogonias undulatus (Linnaeus, 1766) (Perciformes: Sciaenidae), in the South Atlantic Bight off Cow Island (34°38'49″N, 76°33'41″W, type locality) and Figure Eight Island (34°15'48″N, 77°44'27″W), North Carolina, USA, and off Jacksonville Beach (30°08'23″N, 81°20'52″W), Florida, USA. The new species is most easily differentiated from other members of Cardicola Short, 1953 by the combination of having a minute adult body (≤ 1 mm total length) that is 3.1-4.7× longer than wide, widely dispersed ventral tegumental sensory papillae, ~180 tegumental spine rows per side of body, a spheroid anterior sucker that is apparently aspinous, an esophagus that is 38-39% of the body total length, a male genital pore that is anterior to the ootype, a uterus that transitions from ascending to descending portions posterolaterally to the ovary, and a nearly transverse oviducal seminal receptacle. The new species is the second named aporocotylid from a littoral fish of the South Atlantic Bight and the fifth aporocotylid species reported from fishes of the northwestern Atlantic Ocean.     

The distributions of the duplicate oestrogen receptors ER-beta a and ER-beta b in the forebrain of the Atlantic croaker (Micropogonias undulatus): evidence for subfunctionalization after gene duplication

Teleost fishes have three distinct oestrogen receptor (ER) subtypes: ER-alpha, ER-beta a (or ER-gamma) and ER-beta b. ER-beta a and ER-beta b arose from a duplication of an ancestral ER-beta gene early in the teleost lineage. Here, we describe the distribution of the three ER mRNAs in the hypothalamus and cerebellum of the Atlantic croaker to address two issues: the specific functions of multiple ERs in the neuroendocrine system and the evolution and fate of duplicated genes. ER-alpha was detected in nuclei of the preoptic area (POA) and hypothalamus previously shown to possess ER-alphas in teleosts. AcER-beta b, but not ER-beta a, labelling was detected in the magnocellular neurons of the POA, nucleus posterior tuberis, the nucleus recessus posterior and cerebellum. By contrast, acER-beta a, but not ER-beta b, was detected in the dorsal anterior parvocellular POA and suprachiasmatic nucleus. Both ER-betas were found in posterior parvocellular and ventral anterior POA nuclei, the ventral hypothalamus, and periventricular dorsal hypothalamus. The differences we observed in ER subtype mRNA distribution within well-characterized brain nuclei suggest that ER-beta a and ER-beta b have distinct functions in the neuroendocrine control of reproduction and behaviour, and provide evidence that the teleost ER-beta paralogues have partitioned functions of the ancestral ER-beta gene they shared with tetrapods.     

Community ecology of the parasites of adult spot, Leiostomus xanthurus, and Atlantic croaker, Micropogonias undulatus (Sciaenidae) in the Cape Hatteras region

Twenty-three species of metazoan parasites were recorded from adult spot and 26 from adult croaker. Of the 33 parasitic species found, 17 occurred in both spot and croaker. No significant differences in intensity of parasites occurred between sexes of either spot or croaker. All of the parasites had over-dispersed, or clumped, distributions among hosts. Adult spot and croaker collected offshore had much greater species-richness, diversity, and total number of individual parasites than juvenile fishes collected in adjoining estuaries during another study. The juvenile spot and croaker had less time to acquire parasites and inhabited less diverse and more confined habitats in inshore estuaries, which resulted in less diverse parasite communities than offshore fishes. The number of species and diversity of parasites in adult fish was greater in croaker than spot. However, when only gastrointestinal helminths were considered, spot had greater species-richness as well as greater numbers of individual helminths, suggesting that they had a more diverse diet and that they fed on more infected intermediate hosts than croaker. In both adult spot and croaker the mean number of parasitic species was greater than those of freshwater fishes and fewer than those for birds and mammals. The total number of individual parasites was similar to that of freshwater fishes. The opportunistic diet and the migratory habits of both spot and croaker contribute to their diverse parasite faunas. Comparison of adult spot and croaker parasite faunas collected offshore indicated that their respective parasite component communities were distinct and that similar parasite infracommunity variability existed in both hosts and that their communities were not ‘random’ samples, but restricted subsets of the compound community. Although the parasite dominance hierarchy in adults of both species varied slightly between areas and seasons sampled, there appeared to be a predictable dominant species that was accompanied by subordinate, less predictable species. However, the variability in both relative intensities and presence-absence of parasites within communities resulting from their diverse diets make them less predictable than those of other vertebrates with less diverse diets such as the lesser scaup duck and more like those of other marine fishes.     

Induction of oocyte maturation in the white croaker Micropogonias furnieri (Pisces: Sciaenidae) by human chorionic gonadotropin.

The present work aimed to identify the best doses of human chorionic gonadotropin (hCG) needed to induce oocyte maturation of Micropogonias furnieri and to characterize ovarian dynamics during the periovulatory period. Adult M. furnieri females with fully developed ovaries were injected intraperitoneally with four different doses of hCG. The gonadotropin response was succeeded by analyzing morphologically gonadal biopsies and following the postinjection changes in follicle diameter. Oocyte maturation was induced by three doses used: 100, 300, and 500 IU of hCG kg bw-1, and was reached 48 h after treatment with 300 and 500 IU of hCG kg bw-1, and 72 h after treatment with 100 IU of hCG kg bw-1. Concerning ovarian dynamics, only 100 and 300 IU of hCG kg bw-1 mimicked the natural ones which have a synchronic group maturation. In conclusion, the dose mimicking natural ovarian dynamics and inducing oocyte maturation more quickly is 300 IU of hCG kg bw-1.     

There are two forms of androgen binding protein in human testes. Comparison of their protomeric variants with serum testosterone-estradiol binding globulin

To determine how the androgen binding protein in human testes (hABP) is related to the serum protein, testosterone-estradiol binding globulin (hTeBG), both proteins were isolated and compared. The hABP in extracts of human testes was composed of two molecular species based on concanavalin A (ConA)-Sepharose chromatography. Form I hABP did not interact with ConA while Form II hABP bound to ConA and eluted with alpha-methylmannoside. Form I and Form II hABP from five batches of testes were then purified approximately 30,500- and 30,000-fold to apparent homogeneity by high-performance liquid chromatography and compared with hTeBG isolated from human pregnancy serum. Fractionation of both forms of hABP and hTeBG by polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate suggested that the native forms of these proteins were indistinguishable. However, analysis of the purified proteins on sodium dodecyl sulfate-containing polyacrylamide gels indicated that all three were dimers and that each was composed of monomers of at least two sizes which were not present in equimolar concentrations. Two distinctive monomers or protomers of each protein were designated as heavy (H) and light (L) according to their electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels. The H and L protomers of Form I hABP showed apparent molecular weights of 55,000 and 52,000, respectively, in all preparations and were usually present in a 4:5 ratio (H:L). The two components of Form II hABP had apparent molecular weights of 53,000 and 48,000, respectively, and existed in a ratio of approximately 20:1. These two components could not be distinguished in some preparations where Form II hABP migrated as a broad band rather than as distinct protomers. By contrast, hTeBG, which was similar to Form II hABP with respect to ConA binding, always exhibited discrete H and L protomers in a 10:1 ratio. Photolysis of these highly purified proteins with delta 6-[3H]testosterone resulted in specific covalent labeling of their binding sites, confirming that the products identified by silver staining and immunoblotting were indeed steroid binding proteins. The H and L protomers of Form I hABP and hTeBG were separated and examined by peptide mapping using Staphylococcus aureus protease V8 and chymotrypsin. The comparison of the respective fragmentation patterns of protomers indicated that Form I hABP and hTeBG contained distinctive peptides.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of the sex chromosomes of brown trout (Salmo trutta) and their comparison with the corresponding chromosomes in Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss)

Males are the heterogametic sex in salmonid fishes. In brown trout (Salmo trutta) the sex-determining locus, SEX, has been mapped to the end of linkage group BT-28, which corresponds to linkage group AS-8 and chromosome SSA15 in Atlantic salmon (Salmo salar). We set out to identify the sex chromosomes in brown trout. We isolated Atlantic salmon BAC clones containing microsatellite markers that are on BT-28 and also on AS-8, and used these BACs as probes for fluorescent in situ hybridization (FISH) analysis. SEX is located on the short arm of a small subtelocentric/acrocentric chromosome in brown trout, which is consistent with linkage analysis. The acrocentric chromosome SSA15 in Atlantic salmon appears to have arisen by a centric fusion of 2 small acrocentric chromosomes in the common ancestor of Salmo sp. We speculate that the fusion process that produced Atlantic salmon chromosome SSA15 disrupted the ancestral sex-determining locus in the Atlantic salmon lineage, providing the impetus either for the relocation of SEX or selection pressure for a novel sex-determining gene to arise in this species. Thus, the sex-determining genes may differ in Atlantic salmon and brown trout.     

The Escherichia coli PriA helicase has two nucleotide-binding sites differing dramatically in their affinities for nucleotide cofactors. 1. Intrinsic affinities, cooperativities, and base specificity of nucleotide cofactor binding

Interactions of the Escherichia coli PriA helicase with nucleotide cofactors have been studied using the fluorescence titration and analytical ultracentrifugation techniques. Binding of unmodified cofactors was characterized by the fluorescence competition titration method. The obtained data establish that at saturation the PriA helicase binds two nucleotide molecules per protein monomer. This result corroborates with the primary structure of the protein, which contains sequence motifs implicated as putative nucleotide-binding sites. The intrinsic affinities of the binding sites differ by 2-4 orders of magnitude. Thus, the PriA helicase has a strong and a weak nucleotide-binding site. The binding sites differ dramatically in their properties. The strong site is highly specific for adenosine cofactors, while the weak site shows very modest base specificity. The affinities of the strong and weak binding sites for ATP are lower than the affinities for ADP, although both sites have similar affinity for the inorganic phosphate group. Unlike the weak site, the affinity of the strong site profoundly depends on the structure of the phosphate group of the ATP cofactor. Binding of unmodified nucleotides indicates the presence of positive cooperative interactions between bound cofactors (i.e., the existence of communication between the two sites). Magnesium cations are specifically involved in controlling the cofactor affinity for the strong site, while the affinity of the weak site is predominantly determined by interactions between the phosphate group and ribose regions of the cofactor and the protein matrix. The significance of these results for the activities of the PriA helicase is discussed.     

Synthesis of 2-(2,3-dimethoxyphenyl)-4-(aminomethyl)imidazole analogues and their binding affinities for dopamine D2 and D3 receptors

A series of 2-(2,3-dimethoxyphenyl)-4-(aminomethyl)imidazole derivatives was prepared and their affinity for dopamine D₂ and D₃ receptors was measured using in vitro binding assays. Several oxadiazole analogues were also prepared and tested for their affinity for dopamine D₂ and D₃ receptors. The results of receptor binding studies indicated that the incorporation of an imidazole moiety between the phenyl ring and the basic nitrogen did not significantly increase the selectivity for dopamine D₃ receptors, whereas the incorporation of an oxadiazole at the same region resulted in a total loss of affinity for both dopamine receptor subtype binding sites. The most selective compound in this series is 2-(5-bromo-2,3-dimethoxyphenyl)-4-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolinomethyl)imidazole (5i), which has a D₃ receptor affinity of 21 nM and a 7-fold selectivity for D₃ versus D₂ receptors. The binding affinity for σ₁ and σ₂ receptors was also measured, and the results showed that several analogues were selective σ₁ receptor ligands.     

An immunocytochemical method for assaying oestrogen receptors in breast cancers. A comparison with the steroid binding assay

The presence of oestrogen receptors was studied in 105 human breast carcinomas using monoclonal antibodies (Abbott ER-ICA kit). The oestrogen receptors of neoplastic cells were semiquantitatively measured and correlations were made to receptor values determined by a dextran-coated charcoal (DCC) steroid binding assay and to histological grade. Immunoreactive cells were found in about 2/3 of the tumours. Usually only a fraction of the cells within each tumour were immunoreactive, and the staining intensity varied among different cells. In general, well differentiated tumours had a greater proportion of immunoreactive cells than poorly differentiated ones. In most cases (65/98) a good agreement was found between the ER-ICA method and the DCC assay. However, in 33 cases discrepancies were demonstrated.     

The affinities for the two substrate water binding sites in the O(2) evolving complex of photosystem II vary independently during S-state turnover

The first determinations of substrate water binding to the O(2) evolving complex in photosystem II as a complete function of the S states have been made. H(2)(18)O was rapidly injected into spinach thylakoid samples preset in either the S(0), S(1), S(2), or S(3) states, and the rate of (18)O incorporation into the O(2) produced was determined by time-resolved mass spectrometry. For measurements at m/e = 34 (i.e., for the (16)O(18)O product), the rate of (18)O incorporation in all S states shows biphasic kinetics, reflecting the binding of the two substrate water molecules to the catalytic site. The slow phase kinetics yield rate constants at 10 degrees C of 8 +/- 2, 0.021 +/- 0.002, 2.2 +/- 0.3, and 1.9 +/- 0.2 s(-1) for the S(0), S(1), S(2), and S(3) states, respectively, while the fast phase kinetics yield a rate constant of 36.8 +/- 1.9 s(-1) for the S(3) state but remain unresolvable (>100 (s-1)) for the S(0), S(1), and S(2) states. Comparisons of the (18)O exchange rates reveal that the binding affinity for one of the substrate water molecules first increases during the S(0) to S(1) transition, then decreases during the S(1) to S(2) transition, but stays the same during the S(2) to S(3) transition, while the binding affinity for the second substrate water molecule undergoes at least a 5-fold increase on the S(2) to S(3) transition. These findings are discussed in terms of two independent Mn(III) substrate binding sites within the O(2) evolving complex which are separate from the component that accumulates the oxidizing equivalents. One of the Mn(III) sites may only first bind a substrate water molecule during the S(2) to S(3) transition.     

(3H)-estradiol binding by chick liver nuclear extracts: mechanism of increase in binding following estradiol injection.

Specific high affinity binding of [3H]-estradiol by 0.5 M KCl extracts of chick liver nuclei is substantially increased by estradiol injection of the immature chick. The effect is observed shortly after estradiol injection, while the estradiol-induced production of serum phosphoproteins (vitellogenic response) is not detectable until about 24 hr. Cycloheximide given 90 min before estradiol inhibits the increase in nuclear binding for 12-15 hr. At 24-48 hr the levels of nuclear binding are similar to those in the estradiol-treated animals not given cycloheximide, but serum phosphoprotein levels are depressed by about 80% at 48 hr. By 75 hr however the serum of the cycloheximide-treated estrogenized chicks contains about twice as much phosphoprotein as does serum of chicks given estradiol alone. It is suggested that the inhibition of protein synthesis for 12-15 hr delays the vitellogenic response until sufficient levels of nuclear [3H]-estradiol binding protein can be synthesized. A correlation between the levels of nuclear [3H]-estradiol binding at 24 hr and phosphoprotein at 48 hr is shown in a dose-response experiment. In vitro, nafoxidine-HCl (Upjohn 11,100 A) inhibits binding of [3H]-estradiol by the chick liver nuclear extracts. In vivo, a single injection of nafoxidine with estradiol inhibits phosphoprotein production. Injection of nafoxidine alone results in a small but significant increase in [3H]-estradiol binding by nuclear extracts, but it is not estrogenic. A possible interpretation is that nafoxidine transfers low levels of a putative cytosol receptor to the nucleus, but is unable to induce the amplification mechanism required to give the levels of nuclear estradiol-binding protein needed for the vitellogenic response.     

A(1) adenosine receptors in human neutrophils: direct binding and electron microscope visualization.

By occupying specific surface receptors, adenosine and adenosine analogues modulate neutrophil functions; in particular, functional and biochemical studies have shown that A(1) adenosine receptors modulate chemotaxis in response to chemotactic peptides. Until now, the characteristics of the specific agonist binding and the visualization of A(1) receptors in human neutrophils have not been investigated. In the present study, we used the agonist [(3)H] CHA for radioligand binding studies and a CHA-biotin XX probe in order to visualize the A(1) binding sites in human neutrophils, ultrastructurally, by conjugation with colloidal gold-streptavidin. [(3)H] CHA bound A(1) adenosine receptors with selectivity and specificity, although with a low binding capacity. Scatchard analysis showed a Kd value of 1.4 +/- 0.08 nM and a maximum density of binding sites of 7.1 +/- 0.37 fmol/mg of proteins. The good affinity and selectivity of the CHA-biotin XX probe for A(1) adenosine receptors allowed us to visualize them, after conjugation with colloidal gold-streptavidin, as electron-dense gold particles on the neutrophil surface and inside the cell. The internalization of the ligand-receptor complex was followed in a controlled temperature system, and occurred through a receptor-mediated pathway. The kinetics of the intracellular trafficking was fast, taking less than 5 min. These data suggest that the CHA-biotin XX-streptavidin-gold complex is a useful marker for the specific labelling of A(1) binding sites and to follow the intracellular trafficking of these receptors.     

Identification of novel binding partners (annexins) for the cell death signal phosphatidylserine and definition of their recognition motif

Identification and clearance of apoptotic cells prevents the release of harmful cell contents thereby suppressing inflammation and autoimmune reactions. Highly conserved annexins may modulate the phagocytic cell removal by acting as bridging molecules to phosphatidylserine, a characteristic phagocytosis signal of dying cells. In this study five members of the structurally and functionally related annexin family were characterized for their capacity to interact with phosphatidylserine and dying cells. The results showed that AnxA3, AnxA4, AnxA13, and the already described interaction partner AnxA5 can bind to phosphatidylserine and apoptotic cells, whereas AnxA8 lacks this ability. Sequence alignment experiments located the essential amino residues for the recognition of surface exposed phosphatidylserine within the calcium binding motifs common to all annexins. These amino acid residues were missing in the evolutionary young AnxA8 and when they were reintroduced by site directed mutagenesis AnxA8 gains the capability to interact with phosphatidylserine containing liposomes and apoptotic cells. By defining the evolutionary conserved amino acid residues mediating phosphatidylserine binding of annexins we show that the recognition of dying cells represent a common feature of most annexins. Hence, the individual annexin repertoire bound to the cell surface of dying cells may fulfil opsonin-like function in cell death recognition.     

Structure of GABARAP in two conformations: implications for GABA(A) receptor localization and tubulin binding.

GABARAP recognizes and binds the gamma2 subunit of the GABA(A) receptor, interacts with microtubules and the N-ethyl maleimide sensitive factor, and is proposed to function in GABA(A) receptor trafficking and postsynaptic localization. We have determined the crystal structure of human GABARAP at 1.6 A resolution. The structure comprises an N-terminal helical subdomain and a ubiquitin-like C-terminal domain. Structure-based mutational analysis demonstrates that the N-terminal subdomain is responsible for tubulin binding while the C-terminal domain contains the binding site for the GABA(A). A second GABARAP crystal form was determined at 1.9 A resolution and documents that GABARAP can self-associate in a head-to-tail manner. The structural details of this oligomerization reveal how GABARAP can both promote tubulin polymerization and facilitate GABA(A) receptor clustering.