Function of the alpha 1 beta 2 gamma 2S gamma-aminobutyric acid type A receptor is modulated by protein kinase C via multiple phosphorylation sites.

Activation of protein kinase C (PKC) results in down-modulation of the gamma-aminobutyric acid type A (GABAA) receptor. In this study, the recombinant subunit combination alpha 1 beta 2 gamma 2S was expressed in Xenopus oocytes. The resulting channel was shown to be modulated by 2 microM oleoylacetylglycerol or, stereo-specifically, by low concentrations (10 nM) of the phorbol ester 4 beta-phorbol 12-myristate 13-acetate. By site-specific mutagenesis, we altered the serine or threonine residues of consensus phosphorylation sites for PKC in the large, intracellular domain of alpha 1, beta 2, and gamma 2S. Mutant subunits were co-expressed with wild type subunits to yield alpha 1 beta 2 gamma 2S combinations. All of the tested 14 mutations did not affect the level of expression of GABA current. Two of these mutations, Ser-410 in beta 2 and Ser-327 in gamma 2S, resulted in a significant reduction of the effect of the activator of PKC, 4 beta-phorbol 12-myristate 13-acetate, on the GABA current amplitude. Thus, we have identified two single serine residues, Ser-410 in the subunit beta 2 and Ser-327 in gamma 2S, as phosphorylation sites of a PKC endogenous to Xenopus oocytes. Co-expression of the mutant subunits suggests that phosphorylation of both sites is required for a full, PKC-mediated down-regulation of GABA currents.     

An asymmetric contribution to gamma-aminobutyric type A receptor function of a conserved lysine within TM2-3 of alpha1, beta2, and gamma2 subunits

Mutations that impair the expression and/or function of gamma-aminobutyric acid type A (GABAA) receptors can lead to epilepsy. The familial epilepsy gamma2(K289M) mutation affects a basic residue conserved in the TM2-3 linker of most GABAA subunits. We investigated the effect on expression and function of the Lys --> Met mutation in mouse alpha1(K278M), beta2(K274M), and gamma2(K289M) subunits. Compared with cells expressing wild-type and alpha1beta2gamma2(K289M) receptors, cells expressing alpha1(K278M)beta2gamma2 and alpha1beta2(K274M)gamma2 receptors exhibited reduced agonist-evoked current density and reduced GABA potency, with no change in single channel conductance. The low current density of alpha1beta2(K274M)gamma2 receptors coincided with reduced surface expression. By contrast the surface expression of alpha1(K278M)beta2gamma2 receptors was similar to wild-type and alpha1beta2gamma2(K289M) receptors suggesting that the alpha1(K278M) impairs function. In keeping with this interpretation GABA-activated channels mediated by alpha1(K278M)beta2gamma2 receptors had brief open times. To a lesser extent gamma2(K289M) also reduced mean open time, whereas beta2(K274M) had no effect. We used propofol as an alternative GABAA receptor agonist to test whether the functional deficits of mutant subunits were specific to GABA activation. Propofol was less potent as an activator of alpha1(K278M)beta2gamma2 receptors. By contrast, neither beta2(K274M) nor gamma2(K289M) affected the potency of propofol. The beta2(K274M) construct was unique in that it reduced the efficacy of propofol activation relative to GABA. These data suggest that the alpha1 subunit Lys-278 residue plays a pivotal role in channel gating that is not dependent on occupancy of the GABA binding site. Moreover, the conserved TM2-3 loop lysine has an asymmetric function in different GABAA subunits.     

Differential protein mobility of the gamma-aminobutyric acid, type A, receptor alpha and beta subunit channel-lining segments

The gamma-aminobutyric acid, type A (GABAA), receptor ion channel is lined by the second membrane-spanning (M2) segments from each of five homologous subunits that assemble to form the receptor. Gating presumably involves movement of the M2 segments. We assayed protein mobility near the M2 segment extracellular ends by measuring the ability of engineered cysteines to form disulfide bonds and high affinity Zn(2+)-binding sites. Disulfide bonds formed in alpha1beta1E270Cgamma2 but not in alpha1N275Cbeta1gamma2 or alpha1beta1gamma2K285C. Diazepam potentiation and Zn2+ inhibition demonstrated that expressed receptors contained a gamma subunit. Therefore, the disulfide bond in alpha1beta1E270Cgamma2 formed between non-adjacent subunits. In the homologous acetylcholine receptor 4-A resolution structure, the distance between alpha carbon atoms of 20' aligned positions in non-adjacent subunits is approximately 19 A. Because disulfide trapping involves covalent bond formation, it indicates the extent of movement but does not provide an indication of the energetics of protein deformation. Pairs of cysteines can form high affinity Zn(2+)-binding sites whose affinity depends on the energetics of forming a bidentate-binding site. The Zn2+ inhibition IC50 for alpha1beta1E270Cgamma2 was 34 nm. In contrast, it was greater than 100 microM in alpha1N275Cbeta1gamma2 and alpha1beta1gamma2K285C receptors. The high Zn2+ affinity in alpha1beta1E270Cgamma2 implies that this region in the beta subunit has a high protein mobility with a low energy barrier to translational motions that bring the positions into close proximity. The differential mobility of the extracellular ends of the beta and alpha M2 segments may have important implications for GABA-induced conformational changes during channel gating.     

Two novel residues in M2 of the gamma-aminobutyric acid type A receptor affecting gating by GABA and picrotoxin affinity

An amino acid residue was found in M2 of gamma-aminobutyric acid (GABA) type A receptors that has profound effects on the binding of picrotoxin to the receptor and therefore may form part of its binding pocket. In addition, it strongly affects channel gating. The residue is located N-terminally to residues suggested so far to be important for channel gating. Point mutated alpha1beta(3) receptors were expressed in Xenopus oocytes and analyzed using the electrophysiological techniques. Coexpression of the alpha(1) subunit with the mutated beta(3) subunit beta(3)L253F led to spontaneous picrotoxin-sensitive currents in the absence of GABA. Nanomolar concentrations of GABA further promoted channel opening. Upon washout of picrotoxin, a huge transient inward current was observed. The reversal potential of the inward current was indicative of a chloride ion selectivity. The amplitude of the inward current was strongly dependent on the picrotoxin concentration and on the duration of its application. There was more than a 100-fold decrease in picrotoxin affinity. A kinetic model is presented that mimics the gating behavior of the mutant receptor. The point mutation in the neighboring residue beta(3)A252V resulted in receptors that displayed an about 6-fold increased apparent affinity to GABA and an about 10-fold reduced sensitivity to picrotoxin.     

Allosteric effects of chloride ions at the intradimeric alpha1beta1 and alpha2beta2 interfaces of human hemoglobin

The structural transition induced by ligand binding in human hemoglobin encompasses quaternary structure changes at the interfaces between the two alphabeta dimers. In contrast, the interfaces between alpha and beta subunits within the same dimer (i.e., alpha1beta1 and alpha2beta2 interfaces) are structurally invariant. Previous work from this laboratory using NMR spectroscopy has identified four sites at the intradimeric alpha1beta1 and alpha2beta2 interfaces that, although structurally invariant, experience significant changes in the rates of proton exchange upon ligand binding. These sites are Hisalpha103(G10) and Hisalpha122(H5) in each alpha subunit of the hemoglobin tetramer. In the present work, we show that the proton exchange at the Hisalpha103(G10) sites is affected by the interactions of hemoglobin with chloride ions. Increasing concentrations of chloride ions at pH 6.45 and at 37 degrees C enhance the exchange rate of the Hisalpha103(G10) N(epsilon 2) proton. The enhancement is greater in deoxygenated than in ligated hemoglobin. In the framework of the local unfolding model for proton exchange, these results suggest that the structural free energy and/or the proton transfer reactions at the Hisalpha103(G10) sites depend on the concentration of chloride ions. Therefore, the ligand-induced changes at the Hisalpha103(G10) sites are modulated by the allosteric effect of chloride ions on hemoglobin.     

Serine 171, a conserved residue in the gamma-aminobutyric acid type A (GABAA) receptor gamma2 subunit, mediates subunit interaction and cell surface localization

Serine 171 in the GABA(A) receptor gamma2 subunit is highly conserved in the ligand-gated ion channel superfamily. In this paper, we report that mutating serine 171 within gamma2 to glycine or cysteine prevents the interaction of gamma2 with alpha2 and beta1 when these subunits are co-expressed in human embryo kidney 293 cells, resulting in intracellular retention of gamma2. Structure analysis based on a three-dimensional homology model of gamma2 (Ernst, M., Brauchart, D., Boresch, S., and Sieghart, W. (2003) Neuroscience 119, 933-943) reveals that serine 171 may play a critical role in the formation and stabilization of an exposed turn structure that is part of the subunit interaction site. Mutation of serine 171 in the gamma2 subunit could therefore result in alteration of the structure of the subunit interaction site, preventing correct subunit assembly.     

Identification of the cAMP-dependent protein kinase and protein kinase C phosphorylation sites within the major intracellular domains of the beta 1, gamma 2S, and gamma 2L subunits of the gamma-aminobutyric acid type A receptor.

Gamma-aminobutyric acid Type A (GABAA) receptors are the major sites of synaptic inhibition in the central nervous system. These receptors are thought to be pentameric complexes of homologous transmembrane glycoproteins. Molecular cloning has revealed a multiplicity of different GABAA receptor subunits divided into five classes, alpha, beta, gamma, delta, and rho, based on sequence homology. Within the proposed major intracellular domain of these subunits, there are numerous potential consensus sites for protein phosphorylation by a variety of protein kinases. We have used purified fusion proteins of the major intracellular domain of GABAA receptor subunits produced in Escherichia coli to examine the phosphorylation of these subunits by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). The purified fusion protein of the intracellular domain of the beta 1 subunit was an excellent substrate for both PKA and PKC. PKA and PKC phosphorylated the beta 1 subunit fusion protein on serine residues on a single tryptic phosphopeptide. Site-directed mutagenesis of serine 409 in the intracellular domain of the beta 1 subunit to an alanine residue eliminated the phosphorylation of the beta 1 subunit fusion protein by both protein kinases. The purified fusion proteins of the major intracellular domain of the gamma 2S and gamma 2L subunits of the GABAA receptor were rapidly and stoichiometrically phosphorylated by PKC but not by PKA. The phosphorylation of the gamma 2S subunit occurred on serine residues on a single tryptic phosphopeptide. Site-directed mutagenesis of serine 327 of the gamma 2S subunit fusion protein to an alanine residue eliminated the phosphorylation of the gamma 2S fusion protein by PKC. The gamma 2L subunit is an alternatively spliced form of the gamma 2S subunit that differs by the insertion of 8 amino acids (LLRMFSFK) within the major intracellular domain of the gamma 2S subunit. The PKC phosphorylation of the gamma 2L subunit occurred on serine residues on two tryptic phosphopeptides. Site-specific mutagenesis of serine 343 within the 8-amino acid insert to an alanine residue eliminated the PKC phosphorylation of the novel site in the gamma 2L subunit. No phosphorylation of a purified fusion protein of the major intracellular loop of the alpha 1 subunit was observed with either PKA or PKC. These results identify the specific amino acid residues within GABAA receptor subunits that are phosphorylated by PKA and PKC and suggest that protein phosphorylation of these sites may be important in regulating GABAA receptor function.(ABSTRACT TRUNCATED AT 400 WORDS)     

Arg-274 and Leu-277 of the gamma-aminobutyric acid type A receptor alpha 2 subunit define agonist efficacy and potency

Alanine-scanning mutagenesis and the whole cell voltage clamp technique were used to investigate the function of the extracellular loop between the second and third transmembrane domains (TM2-TM3) of the gamma-aminobutyric acid type A receptor (GABA(A)-R). A conserved arginine residue in the TM2-TM3 loop of the GABA(A)-R alpha(2) subunit was mutated to alanine, and the mutant alpha(2)(R274A) was co-expressed with wild-type beta(1) and gamma(2S) subunits in human embryonic kidney (HEK) 293 cells. The GABA EC(50) was increased by about 27-fold in the mutant receptor relative to receptors containing a wild-type alpha(2) subunit. Similarly, the GABA EC(50) at alpha(2)(L277A)beta(1)gamma(2S) and alpha(2)(K279A)beta(1)gamma(2S) GABA(A)-R combinations was increased by 51- and 4-fold, respectively. The alpha(2)(R274A) or alpha(2)(L277A) mutations also reduced the maximal response of piperidine-4-sulfonic acid relative to GABA by converting piperidine-4-sulfonic acid into a weak partial agonist at the GABA(A)-R. Based on these results, we propose that alpha(2)(Arg-274) and alpha(2)(Leu-277) are crucial to the efficient transduction of agonist binding into channel gating at the GABA(A)-R.     

The mechanism of SR95531 inhibition at GABA receptors examined in human alpha1beta1 and alpha1beta1gamma2S receptors

We examined the interaction of GABA and the competitive inhibitor SR95531 at human alpha1beta1gamma2S and alpha1beta1 GABA(A) receptors expressed in Sf9 cells. The efficacy and potency of inhibition depended on the relative timing of the GABA and SR95531 applications. In saturating (10 mM) GABA, the half-inhibitory concentrations of SR95531 (IC50) when coapplied with GABA to alpha1beta1gamma2S or alpha1beta1 receptors were 49 and 210 microM for the peak and 18 and 130 microM for the plateau current, respectively. Our data are explained by an inhibition mechanism in which SR95531 and GABA bind to two sites on the receptor where the binding of GABA allows channel opening but SR95531 does not. The SR95531 affinity for both receptor types was approximately 200 nM and the binding rate was found to be 10-fold faster than that for GABA. The dual binding-site model gives insights into the differential effects of GABA and SR95531 on the peak and plateau currents. The model predicts the effect of SR95531 on GABA currents in the synapse (GABA concentration approximately mM) and at extrasynaptic (GABA concentration < or = microM) sites. The IC50 (50-100 nM) for the synaptic response to SR95531 was insensitive to the GABA affinity of the receptors whereas the IC50 (50-800 nM) for extrasynaptic inhibition correlated with the GABA affinity.     

Tyrosine 62 of the gamma-aminobutyric acid type A receptor beta 2 subunit is an important determinant of high affinity agonist binding

The gamma-aminobutyric acid type A receptor (GABA(A)R) carries both high (K(D) = 10-30 nm) and low (K(D) = 0.1-1.0 microm) affinity binding sites for agonists. We have used site-directed mutagenesis to identify a specific residue in the rat beta2 subunit that is involved in high affinity agonist binding. Tyrosine residues at positions 62 and 74 were mutated to either phenylalanine or serine and the effects on ligand binding and ion channel activation were investigated after the expression of mutant subunits with wild-type alpha1 and gamma2 subunits in tsA201 cells or in Xenopus oocytes. None of the mutations affected [(3)H]Ro15-4513 binding or impaired allosteric interactions between the low affinity GABA and benzodiazepine sites. Although mutations at position 74 had little effect on [(3)H]muscimol binding, the Y62F mutation decreased the affinity of the high affinity [(3)H]muscimol binding sites by approximately 6-fold, and the Y62S mutation led to a loss of detectable high affinity binding sites. After expression in oocytes, the EC(50) values for both muscimol and GABA-induced activation of Y62F and Y62S receptors were increased by 2- and 6-fold compared with the wild-type. We conclude that Tyr-62 of the beta subunit is an important determinant for high affinity agonist binding to the GABA(A) receptor.     

gamma-aminobutyric acid increases the water accessibility of M3 membrane-spanning segment residues in gamma-aminobutyric acid type A receptors

gamma-Aminobutyric acid type A (GABA(A)) receptors are members of the ligand-gated ion channel gene superfamily. Using the substituted cysteine accessibility method, we investigated whether residues in the alpha(1)M3 membrane-spanning segment are water-accessible. Cysteine was substituted, one at a time, for each M3 residue from alpha(1)Ala(291) to alpha(1)Val(307). The ability of these mutants to react with the water-soluble, sulfhydryl-specific reagent pCMBS(-) was assayed electrophysiologically. Cysteines substituted for alpha(1)Ala(291) and alpha(1)Tyr(294) reacted with pCMBS(-) applied both in the presence and in the absence of GABA. Cysteines substituted for alpha(1)Phe(298), alpha(1)Ala(300), alpha(1)Leu(301), and alpha(1)Glu(303) only reacted with pCMBS(-) applied in the presence of GABA. We infer that the pCMBS(-) reactive residues are on the water-accessible surface of the protein and that GABA induces a conformational change that increases the water accessibility of the four M3 residues, possibly by inducing the formation of water-filled crevices that extend into the interior of the protein. Others have shown that mutations of alpha(1)Ala(291), a water-accessible residue, alter volatile anesthetic and ethanol potentiation of GABA-induced currents. Water-filled crevices penetrating into the interior of the membrane-spanning domain may allow anesthetics and alcohol to reach their binding sites and thus may have implications for the mechanisms of action of these agents.     

Subunit-specific coupling between gamma-aminobutyric acid type A and P2X2 receptor channels

ATP and gamma-aminobutyric acid (GABA) are two fast neurotransmitters co-released at central synapses, where they co-activate excitatory P2X and inhibitory GABAA (GABA type A) receptors. We report here that co-activation of P2X2 and various GABAA receptors, co-expressed in Xenopus oocytes, leads to a functional cross-inhibition dependent on GABAA subunit composition. Sequential applications of GABA and ATP revealed that alphabeta- or alphabetagamma-containing GABAA receptors inhibited P2X2 channels, whereas P2X2 channels failed to inhibit gamma-containing GABAA receptors. This functional cross-talk is independent of membrane potential, changes in current direction, and calcium. Non-additive responses observed between cation-selective GABAA and P2X2 receptors further indicate the chloride independence of this process. Overexpression of minigenes encoding either the C-terminal fragment of P2X2 or the intracellular loop of the beta3 subunit disrupted the functional cross-inhibition. We previously demonstrated functional and physical cross-talk between rho1 and P2X2 receptors, which induced a retargeting of rho1 channels to surface clusters when co-expressed in hippocampal neurons (Boue-Grabot, E., Emerit, M. B., Toulme, E., Seguela, P., and Garret, M. (2004) J. Biol. Chem. 279, 6967-6975). Co-expression of P2X2 and chimeric rho1 receptors with the C-terminal sequences of alpha2, beta3, or gamma2 subunits indicated that only rho1-beta3 and P2X2 channels exhibit both functional cross-inhibition in Xenopus oocytes and co-clustering/retargeting in hippocampal neurons. Therefore, the C-terminal domain of P2X2 and the intracellular loop of beta GABAA subunits are required for the functional interaction between ATP- and GABA-gated channels. This gamma subunit-dependent cross-talk may contribute to the regulation of synaptic activity.     

Heterodimerization of gamma-aminobutyric acid B receptor subunits as revealed by the yeast two-hybrid system

Several lines of evidence suggested that the first gamma-aminobutyric acid B receptor to be cloned required an additional factor for functional expression. GABA(B1) was retained within the endoplasmic reticulum and failed to couple to signal transduction pathways on stimulation with agonists. In radioligand binding experiments it was found that although the affinity of antagonists showed a close agreement between rat brain membranes and membranes expressing the cloned receptor, agonist ligands were significantly weaker at recombinant receptors. Using the C-terminal tail as bait, a yeast two-hybrid screen was run against a human brain cDNA library and identified a second receptor, GABA(B2), as a major interacting protein. This interaction was confirmed by coimmunoprecipitation as well as extensive colocalization studies. Coexpression of the two seven-transmembrane proteins generated a fully functional receptor, which was expressed at the cell surface confirming the importance of receptor heterodimerization for GABA(B) receptor activity.     

Constitutive activation of retinoic acid receptor beta2 promoter by orphan nuclear receptor TR2

The orphan nuclear receptor TR2 functions as a constitutive activator for the endogenous retinoic acid receptor beta2 (RAR(beta2)) gene expression in P19 embryonal carcinoma cells and for reporters driven by the RAR(beta2) promoter in COS-1 cells. The activation of RAR(beta2) by TR2 is mediated by the direct repeat-5 (DR5) element located in the RAR(beta2) promoter. Furthermore, cAMP exerts an enhancing effect on the activation of RAR(beta2) by TR2, which is mediated by the cAMP response element located in the 5'-flanking region of the DR5. The constitutive activation function-1 (AF-1) of TR2 is mapped to amino acid residues 10-30 in its N-terminal A segment. A direct molecular interaction occurs between CREMtau and TR2, detected by co-immunoprecipitation, which is mediated by the N-terminal AB segment of TR2. In gel mobility shift assays, TR2 competes with P19 nuclear factor binding to the RAR(beta2) promoter, and TR2 and CREMtau bind simultaneously to this DNA fragment. The role of TR2 in the early events of RA signaling process is discussed.     

Molecular determinants of desensitization and assembly of the chimeric GABA(A) receptor subunits (alpha1/gamma2) and (gamma2/alpha1) in combinations with beta2 and gamma2

Two gamma-aminobutyric acid(A) (GABA(A)) receptor chimeras were designed in order to elucidate the structural requirements for GABA(A) receptor desensitization and assembly. The (alpha1/gamma2) and (gamma2/alpha1) chimeric subunits representing the extracellular N-terminal domain of alpha1 or gamma2 and the remainder of the gamma2 or alpha1 subunits, respectively, were expressed with beta2 and beta2gamma2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (alpha1/gamma2)beta2 and (alpha1/gamma2)beta2gamma2 but not the (gamma2/alpha1)beta2 and (gamma2/alpha1)beta2gamma2 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 (alpha1/gamma2)-containing receptors was pronounced, as opposed to the staining of the (gamma2/alpha1)-containing receptors, which was only slightly higher than background. To explain this, the (alpha1/gamma2) and (gamma2/alpha1) chimeras may act like alpha1 and gamma2 subunits, respectively, indicating that the extracellular N-terminal segment is important for assembly. However, the (alpha1/gamma2) chimeric subunit had characteristics different from the alpha1 subunit, since the (alpha1/gamma2) 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 beta2 or beta2gamma2. Surprisingly, the (alpha1/gamma2)(gamma2/alpha1)beta2 subunit combination did desensitize, indicating that the C-terminal segment of the alpha1 subunit may be important for desensitization. Moreover, desensitization was observed for the (alpha1/gamma2)beta2gamma2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbital and GABA.     

A novel binding site in collagen type III for integrins alpha1beta1 and alpha2beta1

Previously identified high affinity integrin-binding motifs in collagens, GFOGER and GLOGER, are not present in type III collagen. Here, we first characterized the binding of recombinant I domains from integrins alpha(1) and alpha(2) (alpha(1)I and alpha(2)I) to fibrillar collagen types I-III and showed that each I domain bound to the three types of collagens with similar affinities. Using rotary shadowing followed by electron microscopy, we identified a high affinity binding region in human type III collagen recognized by alpha(1)I and alpha(2)I. Examination of the region revealed the presence of two sequences that contain the critical GER motif, GROGER and GAOGER. Collagen-like peptides containing these two motifs were synthesized, and their triple helical nature was confirmed by circular dichroism spectroscopy. Experiments show that the GROGER-containing peptide was able to bind both alpha(1)I and alpha(2)I with high affinity and effectively inhibit the binding of alpha(1)I and alpha(2)I to type III and I collagens, whereas the GAOGER-containing peptide was considerably less effective. Furthermore, the GROGER-containing peptide supported adhesion of human lung fibroblast cells when coated on a culture dish. Thus, we have identified a novel high affinity binding sequence for the collagen-binding integrin I domains.     

Characterization of commercial laminin preparations from human placenta in comparison to recombinant laminins 2 (alpha2beta1gamma1), 8 (alpha4beta1gamma1), 10 (alpha5beta1gamma1).

Laminins, a family of large heterotrimeric (alphabetagamma) proteins, are major components of basement membranes implicated in a variety of cellular functions. Different commercial laminin preparations isolated from human placenta have been widely used in functional studies but their molecular properties are poorly known. In the present study, we characterized several of these preparations by ELISA, silver staining and Western blotting, in comparison to mouse laminin 1 (alpha1beta1gamma1), and recombinant human laminins 2 (alpha2beta1gamma1), 8 (alpha4beta1gamma1) and 10 (alpha5beta1gamma1). The cell migration-promoting activity of different batches was also tested. The placenta laminin preparations differed from one another and consisted of highly fragmented proteins, a mixture of laminin isoforms, and/or contaminating fibronectin. Major functional differences between batches were also observed, reflecting molecular heterogeneity. Previous data obtained in functional studies using these preparations need to be interpreted with caution and may require revision, and future functional studies demand prior molecular characterization of the laminins, particularly their alpha-chain.     

AS160 phosphorylation is associated with activation of alpha2beta2gamma1- but not alpha2beta2gamma3-AMPK trimeric complex in skeletal muscle during exercise in humans

We investigated time- and intensity-dependent effects of exercise on phosphorylation of Akt substrate of 160 kDa (AS160) in human skeletal muscle. Subjects performed cycle exercise for 90 min (67% VO2 peak, n=8), 20 min (80% VO2 peak, n=11), 2 min (110% of peak work rate, n=9), or 30 s (maximal sprint, n=10). Muscle biopsies were obtained before, during, and after exercise. In trial 1, AS160 phosphorylation increased at 60 min (60%, P=0.06) and further at 90 min of exercise (120%, P<0.05). alpha2beta2gamma3-AMP-activated protein kinase (AMPK) activity increased significantly to a steady-state level after 30 min, whereas alpha2beta2gamma1-AMPK activity increased after 60 min of exercise with a further significant increase after 90 min. alpha2beta2gamma1-AMPK activity and AS160 phosphorylation correlated positively (r2=0.55). In exercise trials 2, 3, and 4, alpha2beta2gamma3-AMPK activity but neither AS160 phosphorylation nor alpha2beta2gamma1-AMPK activity increased. Akt Ser473 phosphorylation was unchanged in all trials, whereas Akt Thr308 phosphorylation increased significantly in trial 3 and 4 only. These results show that AS160 is phosphorylated in a time-dependent manner during moderate-intensity exercise and suggest that alpha2beta2gamma1- but not alpha2beta2gamma3-AMPK may act in a pathway responsible for exercise-induced AS160 phosphorylation. Furthermore, we show that AMPK complexes in skeletal muscle are activated differently depending on exercise intensity and duration.