Molecular determinants of desensitization and assembly of the chimeric GABAA receptor subunits (α1/γ2) and (γ2/α1) in combinations with β2 and γ2

Two γ-aminobutyric acid_A (GABA_A) receptor chimeras were designed in order to elucidate the structural requirements for GABA_A receptor desensitization and assembly. The (α1/γ2) and (γ2/α1) chimeric subunits representing the extracellular N-terminal domain of α1 or γ2 and the remainder of the γ2 or α1 subunits, respectively, were expressed with β2 and β2γ2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (α1/γ2)β2 and (α1/γ2)β2γ2 but not the (γ2/α1)β2 and (γ2/α1)β2γ2 subunit combinations formed functional receptor complexes as shown by whole-cell patch–clamp recordings and [³H]muscimol and [³H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (α1/γ2)-containing receptors was pronounced, as opposed to the staining of the (γ2/α1)-containing receptors, which was only slightly higher than background. To explain this, the (α1/γ2) and (γ2/α1) chimeras may act like α1 and γ2 subunits, respectively, indicating that the extracellular N-terminal segment is important for assembly. However, the (α1/γ2) chimeric subunit had characteristics different from the α1 subunit, since the (α1/γ2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch–clamp recordings, which was independent of whether the chimera was expressed in combination with β2 or β2γ2. Surprisingly, the (α1/γ2)(γ2/α1)β2 subunit combination did desensitize, indicating that the C-terminal segment of the α1 subunit may be important for desensitization. Moreover, desensitization was observed for the (α1/γ2)β2γ2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbital and GABA.     

Altered metabolism of bile acids in cholestasis: Determination of 1β- and 6α-hydroxylated metabolites

Trihydroxy and tetrahydroxy bile acid metabolites substituted at the C-1 or C-6 position were studied using the urine, serum and liver tissue from sixteen patients with cholestatic liver diseases. Following extraction, isolation and hydrolysis, bile acids were converted into the dimethylethylsilyl derivatives and assayed by capillary gas chromatography—mass spectrometry. Five 1β-hydroxylated bile acids, viz. 1β,3α,12α-trihydroxy-, 1β,3α,7β-trihydroxy-1, 1β,3α,7α,12α-tetrahydroxy-5β-cholanoic acids and an epimer of the first compound, and two 6α-hydroxylated bile acids, viz. 3α,6α,7α-trihydroxy-, 3α,6α,7α,12α-tetrahydroxy-5β-cholanoic acids, were completely or partially identified. Large amounts of 1β-hydroxylated and 6α-hydroxylated bile acids were found in the urine, whereas only trace amounts were detected in the serum and liver tissue. These findings indicate that altered metabolism, such as 1β- or 6α-hydroxylation of bile acids, is enhanced in cholestasis, and that the resulting hydroxylated metabolites are eliminated in the urine.     

Identification of the NC1 Domain of α3 Chain as Critical for α3α4α5 Type IV Collagen Network Assembly

The network organization of type IV collagen consisting of α3, α4, and α5 chains in the glomerular basement membrane (GBM) is speculated to involve interactions of the triple helical and NC1 domain of individual α-chains, but in vivo evidence is lacking. To specifically address the contribution of the NC1 domain in the GBM collagen network organization, we generated a mouse with specific loss of α3NC1 domain while keeping the triple helical α3 chain intact by connecting it to the human α5NC1 domain. The absence of α3NC1 domain leads to the complete loss of the α4 chain. The α3 collagenous domain is incapable of incorporating the α5 chain, resulting in the impaired organization of the α3α4α5 chain-containing network. Although the α5 chain can assemble with the α1, α2, and α6 chains, such assembly is incapable of functionally replacing the α3α4α5 protomer. This novel approach to explore the assembly type IV collagen in vivo offers novel insights in the specific role of the NC1 domain in the assembly and function of GBM during health and disease.     

Analysis of the α4β1 Integrin–Osteopontin Interaction

The integrin α4β1 is involved in mediating exfiltration of leukocytes from the vasculature. It interacts with a number of proteins up-regulated during the inflammatory response including VCAM-1 and the CS-1 alternatively spliced region of fibronectin. In addition it binds the multifunctional protein osteopontin (OPN), which can act as both a cytokine and an extracellular matrix molecule. Here we map the region of human OPN that supports cell adhesion via α4β1 using GST fusion proteins. We show that α4β1 expressed in J6 cells interacts with intact OPN when the integrin is in a high activation state, and by deletion mapping that the α4β1 binding region in OPN lies between amino acid residues 125 and 168 (aa125–168). This region contains the central RGD motif of OPN, which also interacts with integrins αvβ3, αvβ5, αvβ1, α8β1, and α5β1. Mutating the RGD motif to RAD had no effect on the interaction with α4β1. To define the binding site the region incorporating aa125–168 was divided into 5 overlapping peptides expressed as GST fusion proteins. Two peptides supported adhesion via α4β1, aa132–146, and aa153–168; of these only a synthetic peptide, SVVYGLR (aa162–168), derived from aa153–168 was able to inhibit α4β1 binding to CS-1. These data identify the motif SVVYGLR as a novel peptide inhibitor of α4β1, and the primary α4β1 binding site within OPN.     

Inhibition of adipose differentiation by 9α, 11β-prostaglandin F2α

Prostaglandin F2α (PGF2α) is a potent adipose differentiation inhibitor for the adipogenic cell line 1246 and for adipocyte precursors in primary culture with an ED50 of 3×10⁻⁸ M. In this paper, we examined the effect of several prostaglandins which have structural similarities with PGF2α on the differentiation of 1246 cells and of adipocyte precursors in primary culture. The results show that only 9α,11β-PGF2α is as potent as PGF2α to inhibit differentiation of adipocyte precursors in primary culture and of the adipogenic cell line 1246. In the presence of 9α,11β-PGF2α, the cells remained fibroblast-like, typical of undifferentiated adipocyte precursors. Triglyceride accumulation and increase of specific activity for glycerol-3-phosphate dehydrogenase were inhibited. In addition, mRNA expression of early markers of differentiation such as lipoprotein lipase (LPL) and fatty acid binding protein (FAB) was decreased. The isomer 9β,11α-PGF2α and other PGF2α derivatives were inactive. These results provide new information on the biological activity of 9α,11β-PGF2α as an inhibitor of adipose differentiation and about the structural characteristics of prostaglandins required for maintenance of a high adipose differentiation inhibitory effect.     

The α6β4 Integrin Is a Receptor for both Laminin and Kalinin

Previously, we have established K562 transfectants that express either α6Aβ1 or α6Bβ1 (Kα6A or Kα6B) on their surface. Both cell lines bind to laminin and kalinin after treatment with the β1-stimulatory antibody TS2/16. Here we introduce the full-length β4 cDNA into the α6A- and α6B-expressing K562 cells and selected stably transfected cells. The β4 subunit was expressed on the surface of both transfectants and it formed dimers with the α6A or α6B subunits. Immunoprecipitation and preclearing analyses revealed that both transfectants expressed α6β1, in addition to α6β4. While Kα6A and Kβ6B cells required TS2/16 stimulation for binding to laminin or kalinin, adhesion of the unstimulated β4-transfected Kα6A and Kα6B cells to these matrix components was already substantial. This adhesion was mediated by both α6β1 and α6β4 since it was completely blocked by an α6-specific antibody or by a combination of anti-β1 and anti-β4 antibodies, but only partially by either of these latter two antibodies alone. Adhesion to laminin was completely blocked by an antiserum to laminin fragment E8 as was the adhesion to kalinin by an antibody to kalinin, demonstrating the specificity of adhesion. Both transfectants always adhered more strongly to kalinin than to laminin. Furthermore, binding to kalinin was less well blocked by antibodies to β4 than binding to laminin, indicating that the affinity of α6β4 for kalinin is higher than that for laminin. The fact that α6β1 mediated adhesion without TS2/16 stimulation on the β4-transfected Kα6A and Kα6B cells suggests that some activation of α6β1 had occurred in these cells, even though binding was increased when they were actively stimulated by the antibody TS2/16. Finally, we show that Mn²⁺ induced binding of solubilized α6β4 to matrix containing kalinin, deposited by the murine cell line RAC-11P/SD. This binding was inhibited by the anti-α6 mAb GoH3. Together, these results indicate that both α6β1 and α6β4 are receptors for laminin and kalinin and that there are no differences in ligand specificity between the A and B variants of the α6 subunit when associated with either β1 or β4.     

Isolation of α6β1 integrins from platelets and adherent cells by affinity chromatography on mouse laminin fragment E8 and human laminin pepsin fragment

Ligand affinity chromatography was used to identify receptors on platelets and two adherent cell lines, OVCAR-4 and HBL-100, for the E8 fragment of murine laminin. A complex of two polypeptides (140 and 110 kDa nonreduced) was bound by the E8 affinity columns from all three cell types and was eluted with EDTA. This heterodimeric complex was identified as the α6β1 integrin by immunoprecipitation with specific antibodies against either the α6 or the β1 subunit. The α6β1 integrin did not bind to an affinity column containing fragment P1 originating from a different part of murine laminin which, however, bound the αIIbβ3 integrin from platelets. Furthermore, in immunofluorescence staining, the α6β1 integrin localizes in focal contacts of OVCAR-4 cells attached to laminin and E8 but not to fibronectin substrates. These results, combined with previous antibody inhibition studies, unequivocally identify the α6β1 integrin as a specific receptor for fragment E8. Affinity chromatography of OVCAR-4 and HBL-100 cells on a large pepsin fragment of laminin from human placenta yielded integrin α3β1. When α3β1 was removed from lysates of OVCAR-4 cells by preclearing with an α3-specific monoclonal antibody, α6β1 was able to bind to human laminin as well. Integrin α6β1 on platelets which do not express α3β1 binds directly to human laminin. These results indicate that both α3β1 and α6β1 can act as receptors for human laminin and may interfere by steric hindrance. The α6β4 complex, which is strongly expressed on HBL-100 cells, did not bind to either mouse laminin fragment E8 or human laminin affinity columns.     

Positional Scanning Mutagenesis of α-Conotoxin PeIA Identifies Critical Residues That Confer Potency and Selectivity for α6/α3β2β3 and α3β2 Nicotinic Acetylcholine Receptors

The nicotinic acetylcholine receptor (nAChR) subtype α6β2* (the asterisk denotes the possible presence of additional subunits) has been identified as an important molecular target for the pharmacotherapy of Parkinson disease and nicotine dependence. The α6 subunit is closely related to the α3 subunit, and this presents a problem in designing ligands that discriminate between α6β2* and α3β2* nAChRs. We used positional scanning mutagenesis of α-conotoxin PeIA, which targets both α6β2* and α3β2*, in combination with mutagenesis of the α6 and α3 subunits, to gain molecular insights into the interaction of PeIA with heterologously expressed α6/α3β2β3 and α3β2 receptors. Mutagenesis of PeIA revealed that Asn¹¹ was located in an important position that interacts with the α6 and α3 subunits. Substitution of Asn¹¹ with a positively charged amino acid essentially abolished the activity of PeIA for α3β2 but not for α6/α3β2β3 receptors. These results were used to synthesize a PeIA analog that was >15,000-fold more potent on α6/α3β2β3 than α3β2 receptors. Analogs with an N11R substitution were then used to show a critical interaction between the 11th position of PeIA and Glu¹⁵² of the α6 subunit and Lys¹⁵² of the α3 subunit. The results of these studies provide molecular insights into designing ligands that selectively target α6β2* nAChRs.     

Cross talk between protein kinase CK2 and eukaryotic translation initiation factor eIF2β subunit

The β-subunit of eukaryotic translation initiation factor eIF2 is a substrate and a partner for protein kinase CK2. Surface plasmon resonance analysis shows that the truncated form corresponding to residues 138–333 of eIF2β (eIF2β-CT) interacts with CK2α as efficiently as full length eIF2β, whereas the form corresponding to residues 1–137, which contains the CK2 phosphorylation sites, (eIF2β-NT) does not bind. The use of different mutants and truncated forms of CK2α allowed us to map the basic segment K74–K83 at the beginning of helix αC and residues R191R195K198 in the p+1 loop as the main determinants for the binding to eIF2β-CT of either the isolated CK2α subunit or the CK2 holoenzyme. The presence of eIF2β-CT stimulated the activity of CK2α towards the RRRAADSDDDDD peptide substrate; effect that was not observed with the CK2α K74-77A whose ability to bind to eIF2β-CT is severely impaired. Gel filtration analysis confirmed the ability of CK2α to form complexes with eIF2β-CT, and the contribution of the basic cluster in CK2α (K74–K77) in this association.     

Modulation of Phosphorylation of Tocopherol and Phosphatidylinositol by hTAP1/SEC14L2-Mediated Lipid Exchange

The vitamin E derivative, alpha-tocopheryl phosphate (αTP), is detectable in cultured cells, plasma and tissues in small amounts, suggesting the existence of enzyme(s) with α-tocopherol (αT) kinase activity. Here, we characterize the production of αTP from αT and [γ-³²P]-ATP in primary human coronary artery smooth muscle cells (HCA-SMC) using separation by thin layer chromatography (TLC) and subsequent analysis by Ultra Performance Liquid Chromatography (UPLC). In addition to αT, although to a lower amount, also γT is phosphorylated. In THP-1 monocytes, γTP inhibits cell proliferation and reduces CD36 scavenger receptor expression more potently than αTP. Both αTP and γTP activate the promoter of the human vascular endothelial growth factor (VEGF) gene with similar potency, whereas αT and γT had no significant effect. The recombinant human tocopherol associated protein 1 (hTAP1, hSEC14L2) binds both αT and αTP and stimulates phosphorylation of αT possibly by facilitating its transport and presentation to a putative αT kinase. Recombinant hTAP1 reduces the in vitro activity of the phosphatidylinositol-3-kinase gamma (PI3Kγ) indicating the formation of a stalled/inactive hTAP1/PI3Kγ heterodimer. The addition of αT, βT, γT, δT or αTP differentially stimulates PI3Kγ, suggesting facilitated egress of sequestered PI from hTAP1 to the enzyme. It is suggested that the continuous competitive exchange of different lipophilic ligands in hTAPs with cell enzymes and membranes may be a way to make these lipophiles more accessible as substrates for enzymes and as components of specific membrane domains.     

Mapping of the α4 subunit gene (GABRA4) to human chromosome 4 defines an α2—α4—β1—γ1 gene cluster: further evidence that modern GABAA receptor gene clusters are derived from an ancestral cluster

We demonstrated previously that an α₁—β₂—γ₂ gene cluster of the γ-aminobutyric acid (GABA_A) receptor is located on human chromosome 5q34–q35 and that an ancestral α—β—γ gene cluster probably spawned clusters on chromosomes 4, 5, and 15. Here, we report that the α₄ gene (GABRA4) maps to human chromosome 4p14–q12, defining a cluster comprising the α₂, α₄, β₁, and γ₁ genes. The existence of an α₂—α₄—β₁—γ₁ cluster on chromosome 4 and an α₁—α₆—β₂—γ₂ cluster on chromosome 5 provides further evidence that the number of ancestral GABA_A receptor subunit genes has been expanded by duplication within an ancestral gene cluster. Moreover, if duplication of the α gene occurred before duplication of the ancestral gene cluster, then a heretofore undiscovered subtype of α subunit should be located on human chromosome 15q11–q13 within an α₅—α_x—β₃—γ₃ gene cluster at the locus for Angelman and Prader—Willi syndromes.     

Simultaneous determination of 6β- and 6α-hydroxycortisols and 6β-hydroxycortisone in human urine by stable isotope dilution mass spectrometry

A capillary gas chromatographic–mass spectrometric method for the simultaneous determination of 6β-hydroxycortisol (6β-OHF, 6β,11β,17α,21-tetrahydroxypregn-4-ene-3,20-dione), 6α-hydroxycortisol (6α-OHF, 6α,11β,17α,21-tetrahydroxypregn-4-ene-3,20-dione) and 6β-hydroxycortisone (6β-OHE, 6β,17α,21-trihydroxypregn-4-ene-3,11,20-trione) in human urine is described. Deuterium-labelled compounds, 6β-[1,1,19,19,19-²H₅]OHF (6β-OHF-d₅), 6α-[1,1,19,19,19-²H₅]OHF (6α-OHF-d₅) and 6β-[1,1,19,19,19-²H₅]OHE (6β-OHE-d₅) were used as internal standards. Quantitation was carried out by selected-ion monitoring of the characteristic fragment ions ([M-31]⁺) of the methoxime–trimethylsilyl (MO–TMS) derivatives of 6β-OHF, 6α-OHF and 6β-OHE. The sensitivity, specificity, precision and accuracy of the method were demonstrated to be satisfactory for measuring 6β-OHF, 6α-OHF and 6β-OHE in human urine.     

Stable isotope dilution analysis of human urinary metabolites of 17α-methyltestosterone

A method based on gas chromatography–mass spectrometry–selected-ion monitoring was developed to measure the main metabolites of 17α-methyltestosterone, 17α-methyl-5α-androstan-3α,17β-diol and 17α-methyl-5β-androstan-3α,17β-diol, in human urine. 17α-Methyl-[²H₃]-5α-androstan-3α,17β-diol and 17α-methyl-[²H₃]-5β-androstan-3α,17β-diol were used as internal standards. The methods involved purification using a Sep-Pak C₁₈ cartridge, hydrolysis by β-glucuronidase from Ampullaria and derivatization with N-methyl-N-trimethylsilyl-trifluoroacetamide/dithioerythriol/ammonium iodide. Quantitation was achieved by selected-ion monitoring of the characteristic fragment ions ([(M+H)−2×TMSOH]⁺) of the di-TMS derivatives on the chemical ionization mode. The method provides a specific, sensitive and reliable technique to determine the urine levels of 17α-methyl-5α-androstan-3α,17β-diol and 17α-methyl-5β-androstan-3α,17β-diol, and can be applied to pharmacokinetic studies of 17α-methyltestosterone.     

Simultaneous determination of α-tocopheryl acetate and tocopherols in aquatic organisms and fish feed

In aquaculture, α-tocopheryl acetate (α-TA) is the main source of vitamin E used to fortify fish feed. α-TA in fish is often determined indirectly, i.e., by alkaline hydrolysis, followed by quantitation of “total α-tocopherol” (α-T) and subtraction of the natively present α-T. The aim of this study was to develop an HPLC method for the simultaneous quantitative determination of α-TA and free tocopherols in aquatic organisms and fish feed. The assay consists of a simple extraction with methanol containing butylhydroxytoluene (BHT) as an antioxidant, followed by reversed-phase chromatography with consecutive UV and fluorescence detection of α-TA and tocopherols, respectively. The peak of the internal standard tocol in the fluorescence trace was used for quantitation. Linearity was achieved over the range of 0.2 to 4.2 μg α-TA per ml extract of Artemia nauplii, which would correspond to 30.7 to 614.4 μg/g dry mass. The within-run coefficient of variation was 1.9% at a level of 310 μg/g dry mass. The recovery of α-TA ranged from 97.7 to 100.8% (concentration=2.1 and 20.5 μg/ml, n=6). The detection limit was about 7 ng and the quantification limit on spiked samples was 0.2 μg/ml. This method was routinely applied to determine α-TA and α-, γ- and δ-tocopherol (α-T, γ-T, δ-T) simultaneously in Artemia, fish feed, shrimp eggs and various other aquatic organisms.     

The Role of Extracellular and Cytoplasmic Splice Domains of α7-Integrin in Cell Adhesion and Migration on Laminins

The major laminin-binding integrin of skeletal, smooth, and heart muscle is α7β1-integrin, which is structurally related to α6β1. It occurs in three cytoplasmic splice variants (α7A, -B, and -C) and two extracellular forms (X1 and X2) which are developmentally regulated and differentially expressed in skeletal muscle. Previously, we have shown that ectopic expression of the α7β-integrin splice variant in nonmotile HEK293 cells specifically induced cell locomotion on laminin-1 but not on fibronectin. To investigate the specificity and the mechanism of the α7-mediated cell motility, we expressed the three α7-chain cytoplasmic splice variants, as well as α6A- and α6B-integrin subunits in HEK293 cells. Here we show that all three α7 splice variants (containing the X2 domain), as well as α6A and α6B, promote cell attachment and stimulate cell motility on laminin-1 and its E8 fragment. Deletion of the cytoplasmic domain (excluding the GFFKR consensus sequence) from α7B resulted in a loss of the motility-enhancing effect. On laminin-2/4 (merosin), the predominant isoform in mature skeletal muscle, only α7-expressing cells showed enhanced motility, whereas cells transfected with α6A and α6B neither attached nor migrated on laminin-2. Adhesion of α7-expressing cells to both laminin-1 and laminin-2 was specifically inhibited by a new monoclonal antibody (6A11) specific for α7. Expression of the two extracellular splice variants α7X1 and α7X2 in HEK293 cells conferred different motilities on laminin isoforms: Whereas α7X2B promoted cell migration on both laminin-1 and laminin-2, α7X1B supported motility only on laminin-2 and not on laminin-1, although both X1 and X2 splice variants revealed similar adhesion rates to laminin-1 and -2. Fluorescence-activated cell sorter analysis revealed a dramatic reduction of surface expression of α6-integrin subunits after α7A or -B transfection; also, surface expression of α1-, α3-, and α5-integrins was significantly reduced. These results demonstrate selective responses of α6- and α7-integrins and of the α7 splice variants to laminin-1 and -2 and indicate differential roles in laminin-controlled cell adhesion and migration.     

Moieties of Complement iC3b Recognized by the I-domain of Integrin αXβ2

Complement fragment iC3b serves as a major opsonin for facilitating phagocytosis via its interaction with complement receptors CR3 and CR4, also known by their leukocyte integrin family names, αMβ2 and αXβ2, respectively. Although there is general agreement that iC3b binds to the αM and αX I-domains of the respective β2-integrins, much less is known regarding the regions of iC3b contributing to the αX I-domain binding. In this study, using recombinant αX I-domain, as well as recombinant fragments of iC3b as candidate binding partners, we have identified two distinct binding moieties of iC3b for the αX I-domain. They are the C3 convertase-generated N-terminal segment of the C3b α’-chain (α’NT) and the factor I cleavage-generated N-terminal segment in the CUBf region of α-chain. Additionally, we have found that the CUBf segment is a novel binding moiety of iC3b for the αM I-domain. The CUBf segment shows about a 2-fold higher binding activity than the α’NT for αX I-domain. We also have shown the involvement of crucial acidic residues on the iC3b side of the interface and basic residues on the I-domain side.     

Glycoprofile of the different cell types present in the mucosa of the horse guttural pouches

Histochemical characterization of the equine guttural pouches was performed using lectins combined with sialidase digestion and deglycosylation pre-treatments.The goblet cells contained O- and N-linked oligosaccharides with α-Fuc, GlcNAc moieties whereas β-GalNAc, β-Gal-(1–3)-GalNAc, β-Gal-(1–4)-GlcNAc and α-Gal residues belonged only to O-linked glycoproteins. The acinar and ductal cells expressed α-Man/α-Glc in N-linked oligosaccharides, GlcNAc in both O- and N-glycoproteins and β-GalNAc, β-Gal-(1–3)-GalNAc, β-Gal-(1–4)-GlcNAc and α-Gal residues included in O-linked glycoproteins. The Golgi area of the epithelial lining expressed α-Fuc in O-linked glycoproteins, internal GlcNAc in N-linked glycoproteins and large amounts of sialic acid residues linked to subterminal β-GalNAc, Galβ1,4GlcNAc and Galβ1,3GalNAc. High amounts of sulpho-carbohydrates and of sialic acids (α2,3–6), linked to-α/β-Gal and sialic acids (α2–6) linked to β-GalNAc, were also demonstrated.Such diversity of the mucin saccharide residues may be implicated in the binding of macromolecules such as those of bacterial or viral etiology, thus playing a role in the organism's host-defense mechanism in the guttural pouches.     

Key Residues in the Nicotinic Acetylcholine Receptor β2 Subunit Contribute to α-Conotoxin LvIA Binding

α-Conotoxin LvIA (α-CTx LvIA) is a small peptide from the venom of the carnivorous marine gastropod Conus lividus and is the most selective inhibitor of α3β2 nicotinic acetylcholine receptors (nAChRs) known to date. It can distinguish the α3β2 nAChR subtype from the α6β2* (* indicates the other subunit) and α3β4 nAChR subtypes. In this study, we performed mutational studies to assess the influence of residues of the β2 subunit versus those of the β4 subunit on the binding of α-CTx LvIA. Although two β2 mutations, α3β2[F119Q] and α3β2[T59K], strongly enhanced the affinity of LvIA, the β2 mutation α3β2[V111I] substantially reduced the binding of LvIA. Increased activity of LvIA was also observed when the β2-T59L mutant was combined with the α3 subunit. There were no significant difference in inhibition of α3β2[T59I], α3β2[Q34A], and α3β2[K79A] nAChRs when compared with wild-type α3β2 nAChR. α-CTx LvIA displayed slower off-rate kinetics at α3β2[F119Q] and α3β2[T59K] than at the wild-type receptor, with the latter mutant having the most pronounced effect. Taken together, these data provide evidence that the β2 subunit contributes to α-CTx LvIA binding and selectivity. The results demonstrate that Val¹¹¹ is critical and facilitates LvIA binding; this position has not previously been identified as important to binding of other 4/7 framework α-conotoxins. Thr⁵⁹ and Phe¹¹⁹ of the β2 subunit appear to interfere with LvIA binding, and their replacement by the corresponding residues of the β4 subunit leads to increased affinity.     

Integrin α3β1 regulates kidney collecting duct development via TRAF6-dependent K63-linked polyubiquitination of Akt

The collecting system of the kidney develops from the ureteric bud (UB), which undergoes branching morphogenesis, a process regulated by multiple factors, including integrin–extracellular matrix interactions. The laminin (LM)-binding integrin α3β1 is crucial for this developmental program; however, the LM types and LM/integrin α3β1–dependent signaling pathways are poorly defined. We show that α3 chain–containing LMs promote normal UB branching morphogenesis and that LM-332 is a better substrate than LM-511 for stimulating integrin α3β1–dependent collecting duct cell functions. We demonstrate that integrin α3β1–mediated cell adhesion to LM-332 modulates Akt activation in the developing collecting system and that Akt activation is PI3K independent but requires decreased PTEN activity and K63-linked polyubiquitination. We identified the ubiquitin-modifying enzyme TRAF6 as an interactor with the integrin β1 subunit and regulator of integrin α3β1–dependent Akt activation. Finally, we established that the developmental defects of TRAF6- and integrin α3–null mouse kidneys are similar. Thus K63-linked polyubiquitination plays a previously unrecognized role in integrin α3β1–dependent cell signaling required for UB development and may represent a novel mechanism whereby integrins regulate signaling pathways.