Biomedical subjects

Antony A Boucard

Publications and source records attributed to Antony A Boucard.

7 recordsLinked to original sources

PubMed2006

Gene selection, alternative splicing, and post-translational processing regulate neuroligin selectivity for beta-neurexins.

Neuroligins 1-4 are postsynaptic transmembrane proteins capable of initiating presynaptic maturation via interactions with beta-neurexin. Both neuroligins and beta-neurexins have alternatively spliced inserts in their extracellular domains. Using analytical ultracentrifugation, we determined that the extracellular domains of the neuroligins sediment as dimers, whereas the extracellular domains of the beta-neurexins appear monomeric. Sedimentation velocity experiments of titrated stoichiometry ratios of beta-neurexin and neuroligin suggested a 2:2 complex formation. The recognition properties of individual neuroligins toward beta-neurexin-1 (NX1beta), along with the influence of their splice inserts, were explored by surface plasmon resonance and affinity chromatography. Different neuroligins display a range of NX1beta affinities spanning more than 2 orders of magnitude. Whereas splice insert 4 in beta-neurexin appears to act only as a modulator of the neuroligin/beta-neurexin association, splice insert B in neuroligin-1 (NL1) is the key element regulating the NL1/NX1beta binding. Our data indicate that gene selection, mRNA splicing, and post-translational modifications combine to give rise to a controlled neuroligin recognition code with a rank ordering of affinities for particular neurexins that is conserved for the neuroligins across mammalian species.

Alternative Splicing↗

PubMed2005

A splice code for trans-synaptic cell adhesion mediated by binding of neuroligin 1 to alpha- and beta-neurexins.

Previous studies suggested that postsynaptic neuroligins form a trans-synaptic complex with presynaptic beta-neurexins, but not with presynaptic alpha-neurexins. Unexpectedly, we now find that neuroligins also bind alpha-neurexins and that alpha- and beta-neurexin binding by neuroligin 1 is regulated by alternative splicing of neuroligin 1 (at splice site B) and of neurexins (at splice site 4). In neuroligin 1, splice site B is a master switch that determines alpha-neurexin binding but leaves beta-neurexin binding largely unaffected, whereas alternative splicing of neurexins modulates neuroligin binding. Moreover, neuroligin 1 splice variants with distinct neurexin binding properties differentially regulate synaptogenesis: neuroligin 1 that binds only beta-neurexins potently stimulates synapse formation, whereas neuroligin 1 that binds to both alpha- and beta-neurexins more effectively promotes synapse expansion. These findings suggest that neuroligin binding to alpha- and beta-neurexins mediates trans-synaptic cell adhesion but has distinct effects on synapse formation, indicating that expression of different neuroligin and neurexin isoforms specifies a trans-synaptic signaling code.

Alternative Splicing↗

PubMed2004

Analysis of the third transmembrane domain of the human type 1 angiotensin II receptor by cysteine scanning mutagenesis.

Activation of G protein-coupled receptors by agonists involves significant movement of transmembrane domains (TMD) following agonist binding. The underlying structural mechanism by which receptor activation takes place is largely unknown but can be inferred by detecting variability within the environment of the ligand-binding pocket, which is a water-accessible crevice surrounded by the seven TMD helices. Using the substituted-cysteine accessibility method, we identified the residues within the third TMD of the wild-type angiotensin II (AT1) receptor that contribute to the formation of the binding site pocket. Each residue within the Ile103-Tyr127 region was mutated one at a time to a cysteine. Treating the A104C, N111C, and L112C mutant receptors with the charged sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA) strongly inhibited ligand binding, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD3 reporter cysteines engineered in a constitutively active AT1 receptor. Indeed, two additional mutants (S109C and V116C) were found to be sensitive to MTSEA treatment. Our results suggest that constitutive activation of the AT1 receptor causes a minor counterclockwise rotation of TMD3, thereby exposing residues, which are not present in the inactive state, to the binding pocket. This pattern of accessibility of residues in the TMD3 of the AT1 receptor parallels that of homologous residues in rhodopsin. This study identified key elements of TMD3 that contribute to the activation of class A G protein-coupled receptors through structural rearrangements.

Amino Acid Sequence↗

PubMed2003

Constitutive activation of the angiotensin II type 1 receptor alters the spatial proximity of transmembrane 7 to the ligand-binding pocket.

Activation of G protein-coupled receptors by agonists involves significant movement of transmembrane domains (TM) following binding of agonist. The underlying structural mechanism by which receptor activation takes place is largely unknown but can be inferred by detecting variability within the environment of the ligand-binding pocket, which constitutes a water-accessible crevice surrounded by the seven TM helices. Using the substituted cysteine accessibility method, we initially identified those residues within the seventh transmembrane domain (TM7) of wild type angiotensin II type 1 (AT1) receptor that contribute to forming the binding site pocket. We have substituted successively TM7 residues ranging from Ile276 to Tyr302 to cysteine. Treatment of A277C, V280C, T282C, A283C, I286C, A291C, and F301C mutant receptors with the charged sulfhydryl-specific alkylating agent MTSEA significantly inhibited ligand binding, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was greatly reduced for TM7 reporter cysteines engineered in a constitutively active mutant of the AT1 receptor. Our data suggest that upon activation, TM7 of the AT1 receptor goes through a pattern of helical movements that results in its distancing from the binding pocket per se. These studies support accumulating evidence whereby elements of TM7 of class A GPCRs promote activation of the receptor through structural rearrangements.

Amino Acid Sequence↗

PubMed2003

Photolabelling the rat urotensin II/GPR14 receptor identifies a ligand-binding site in the fourth transmembrane domain.

A urotensin II (U-II) peptide analogue containing the photoreactive p -benzoyl-L-phenylalanine (Bz-Phe) in the sixth position was used to identify ligand-binding sites of the rat U-II receptor, also known as GPR14. [Bz-Phe(6)]U-II bound the receptor expressed in COS-7 cells with high affinity (IC(50) 0.7 nM) and was as potent as U-II in the agonist-induced production of inositol phosphate. Photolabelling of the U-II receptor with (125)I-[Bz-Phe(6)]U-II resulted in the specific formation of a glycosylated (125)I-[Bz-Phe(6)]U-II-U-II receptor complex of 60 kDa. Digestion of the 60 kDa complex with endoproteinase Glu-C generated a fragment of 17 kDa circumscribing the labelled fragment to residues 148-286. Digestion of the ligand-receptor complex with endoproteinase Arg-C produced a short peptide of 4 kDa corresponding to fragments 125-148, 167-192 or 210-233. CNBr treatment of the endoproteinase-Glu-C and -Arg-C fragments yielded 2 kDa fragments, defining the labelling site to methionine residues 184/185 of the fourth transmembrane domain. Photolabelling of two mutant receptors, M184L/M185L and M184A/M185A, led to a significant decrease in the overall yield of covalent labelling. Taken together, our results indicate that position 6 of U-II normally occupied by phenylalanine would interact with Met(184) and/or Met(185) of the fourth transmembrane domain of the U-II receptor. This information should be of significant value in the study of the interactions between U-II and its cognate receptor.

Amidohydrolases↗

PubMed2002

Residues 293 and 294 are ligand contact points of the human angiotensin type 1 receptor.

The human angiotensin II type 1 receptor (hAT(1)) was photolabeled with a high-affinity radiolabeled photoreactive analogue of AngII, (125)I-[Sar(1), Val(5), p-Benzoyl-L-phenylalanine(8)]AngII ((125)I-[Sar(1),Bpa(8)]AngII). Chemical cleavage with CNBr produced a 7 kDa fragment (285-334) of the C-terminal portion of the hAT(1). Manual Edman radiosequencing of photolabeled, per-acetylated, and CNBr-fragmented receptor showed that ligand incorporation occurred through Phe(293) and Asn(294) within the seventh transmembrane domain of the hAT(1). Receptor mutants with Met introduced at the presumed contact residues, F293M and N294M, were photolabeled and then digested with CNBr. SDS-PAGE analysis of those digested mutant receptors confirmed the contact positions 293 and 294 through ligand release induced by CNBr digestion. Additional receptor mutants with Met residues introduced into the N- and C-terminal proximity of those residues 293 and 294 of the hAT(1) produced, upon photolabeling and CNBr digestion, fragmentation patterns compatible only with the above contact residues. These data indicate that the C-terminal residue of AngII interacts with residues 293 and 294 of the seventh transmembrane domain of the human AT(1) receptor. Taking into account a second receptor-ligand contact at the second extracellular loop and residue 3 of AngII (Boucard, A. A., Wilkes, B. C., Laporte, S. A., Escher, E., Guillemette, G., and Leduc, R. (2000) Biochemistry 39, 9662-70) the Ang II molecule must adopt an extended structure in the AngII binding pocket.

Amino Acid Substitution↗

PubMedDate not supplied

Methionine proximity assay, a novel method for exploring peptide ligand-receptor interaction.

Probing G-protein coupled receptor (GPCR) structures is a priority in the functional and structural understanding of GPCRs. In the past, we have used several approaches around photoaffinity labeling in order to establish contact points between peptide ligands and their cognate receptors. Such contact points are helpful to build reality based molecular models of GPCRs and to elucidate their activation mechanisms. Most studies of peptidergic GPCRs have been done with photolabeling peptides containing the benzophenone moiety as a reputedly non-selective probe. However our recent results are now showing that p-benzoylphenylalanine (Bpa) has some selectivity for Met residues in the receptor protein, reducing the accuracy of this method. Turning a problem into an asset, modified analogues of Bpa, e.g. p,p'-nitrobenzoylphenylalanine (NO2Bpa), display increased selectivity for such Met residues. It means a photoprobe containing such modified benzophenone-moieties does not label a receptor protein unless a Met residue is in the immediate vicinity. This unique property allows us to propose and show the feasibility and utility of a new method for scanning the contact areas of peptidergic GPCRs, the Methionine Proximity Assay (MPA). Putative contact residues of the receptor are exchanged to Met residues by site-directed mutagenesis and are subjected to photoaffinity labeling with such modified benzophenone-containing peptides. Successful incorporation indicates physical proximity of those residues. This principle is established and explored with benzophenone-containing analogues of angiotensin II and the two known human angiotensin II receptors AT1 and AT2, determining contact points in both receptors. This approach has several important advantages over other scanning approaches, e.g., the SCAM procedure, since the MPA-method can be used in the hydrophobic core of receptors.

Angiotensin II↗