Search PubMed⌕ Search

Biomedical subjects

Y Saad

Publications and source records attributed to Y Saad.

At least 19 recordsLinked to original sources
PubMed1997

Transducin-alpha C-terminal peptide binding site consists of C-D and E-F loops of rhodopsin.

The binding of heterotrimeric GTP-binding proteins (G-proteins) to serpentine receptors involves several independent contacts. We have deduced the points of interaction between mutant bovine rhodopsins and alphat-(340-350), a peptide corresponding to the C terminus of the alpha subunit (alphat) of bovine retinal G-protein, transducin. Direct binding of alphat-(340-350) to rhodopsin stabilizes the activated metarhodopsin II state (M II), consequently uncoupling the rhodopsin-transducin interaction. This peptide action requires two segments on the cytoplasmic domain of rhodopsin: the Tyr136-Val137-Val138-Val139 sequence on the C-D loop and the Glu247-Lys248-Glu249-Val250-Thr251 sequence on the E-F loop. We propose that a tertiary interaction of these two loop regions forms a pocket for binding the alphat C terminus of the transducin during light transduction in vivo. In most G-proteins, the C termini of alpha subunits are important for interaction with receptors, and, in several serpentine receptors, regions similar to those in rhodopsin are essential for G-protein activation, indicating that the interaction described here may be a generally applicable mode of G-protein binding in signal transduction.

Amino Acid Sequence↗
PubMed1996

The active state of the AT1 angiotensin receptor is generated by angiotensin II induction.

In the current model of receptor activation, the given hormone is not involved in the conversion of the inactive receptor (R) to the fully active state (R*). Rather, it preferentially selects the activated receptor conformation, thereby shifting the equilibrium toward R*. The hormone angiotensin II (Ang II) contains two residues, Tyr4 and Phe8, that are essential for agonism. We show that the conserved Asn111 in transmembrane helix III of the AT1 angiotensin receptor directly interacts with the Tyr4 side chain. A decrease in the size of the Asn111 side chain induces an intermediate activated receptor conformation (R'). The Ang II analogue [Sar1,Ile4,Ile8]Ang II fully activates the N111G mutant, indicating that either the transition from R' to R* or the stabilization of the R* state requires binding by Ang II but not its Tyr4 and Phe8 side chains. In contrast, [Sar1,Ile4,Ile8]Ang II binds to but does not activate the wild-type AT1 receptor (R), suggesting that in the wild-type receptor spontaneous occurrence of R' and R* states is rare. Thus, Ang II through interactions involving Tyr4 and Phe8 induces a transition from R to R' and through unspecified interactions induces transition from R' to R* states rather than stabilizing the spontaneously generated R* state by "conformational, selection".

Amino Acid Sequence↗
PubMed1995

Interaction of Phe8 of angiotensin II with Lys199 and His256 of AT1 receptor in agonist activation.

The acidic pharmacophores of selective ligands bind to Lys199 and His256 of the AT1 receptor (Noda, K., Saad, Y., Kinoshita, A., Boyle, T. P., Graham, R. M., Husain, A., and Karnik, S. (1995) J. Biol. Chem. 270, 2284-2289). In this report we examine how interactions between these residues and agonists activate inositol phosphate production in transiently transfected COS-1 cells. [Sar1] angiotensin (Ang II) II and [Sar1]Ang II-amide stimulated a 5-fold inositol phosphate response from wild-type AT1 receptor. The peptide antagonist [Sar1,Ile8]Ang II and the non-peptide agonist L-162,313 produced a partial but saturating response. Stimulation of wild-type receptor by [Sar1]Ang II-amide and the mutant K199Q and K199A receptors by [Sar1]Ang II demonstrates that AT1 receptor activation is not critically dependent on the ion-pairing of the alpha-COOH group of Ang II with Lys199. The mutation of His256 produced diminished inositol phosphate response without commensurate change in binding affinity of ligands. The His256 side chain is critical for maximal activation of the AT1 receptor, although isosteric Gln substitution is sufficient for preserving the affinity for Phe8-substituted analogues of [Sar1]Ang II. Therefore, AT1 receptor activation requires interaction of Phe8 side chain of Ang II with His256, which is achieved by docking the alpha-COOH group of Phe8 to Lys199. Furthermore, non-peptide agonists interact with Lys199 and His256 in a similar fashion.

Angiotensin I↗
PubMed1995

The docking of Arg2 of angiotensin II with Asp281 of AT1 receptor is essential for full agonism.

The structural model of AT1 angiotensin receptor contains seven-transmembrane alpha-helices with three interhelical loops on either side of the membrane. The angiotensin II binding pocket within the receptor is not clearly defined. We showed earlier that Lys199 in transmembrane-helix-5 of the AT1 receptor binds the COOH-terminal alpha-carboxyl group of angiotensin II (Noda, K., Saad, Y., Kinoshita, A., Boyle, T. P., Graham, R. M., Husain, A., and Karnik, S. S. (1995) J. Biol. Chem. 270, 2284-2289). We now show that His183 and Asp281, both located in the extracellular domain of the AT1 receptor, are involved in binding the NH2-terminal Asp1 and Arg2 residues of angiotensin II, respectively. The Asp1/His183 interaction appears to be weak and is unlikely to be important for agonism. But the loss of Arg2/Asp281 interaction leads to partial agonism of the receptor. The action of non-peptide agonists is not affected by Asp281 mutations. These results suggest that several independent interactions between angiotensin II and AT1 receptor are necessary for full agonism. Since L-162,313 the non-peptide agonist of the AT1 receptor is a partial agonist that does not make contact with Asp281, we speculate that the degree of agonism may be increased if it is redesigned to make contacts with Asp281.

Angiotensin II↗
PubMed1995

Tetrazole and carboxylate groups of angiotensin receptor antagonists bind to the same subsite by different mechanisms.

To identify specific interactions between either the tetrazole or carboxylate pharmacophores of non-peptide antagonists and the rat AT1 receptor, 6 basic residues were examined by site-directed mutagenesis. Three of the mutants (H183Q, H256Q, and H272Q) appeared to be like wild type. Lys102 and Arg167 mutants displayed reduced binding of the non-peptide antagonist losartan. Examination of their properties employing group-specific angiotensin II analogues indicated that their effects on binding were indirect. Interestingly, the affinity of losartan was not altered by a K199Q mutation, but the same mutation reduced the affinity of angiotensin II, the antagonist [Sar1,Ile8]angiotensin II, and several carboxylate analogues of losartan. An Ala199 substitution reduced the affinity of peptide analogues to a larger extent as compared to the affinity of losartan. Thus, the crucial acidic pharmacophores of angiotensin and losartan appear to occupy the same space within the receptor pocket, but the protonated amino group of Lys199 is not essential for binding the tetrazole anion. The binding of the tetrazole moiety with the AT1 receptor involves multiple contacts with residues such as Lys199 and His256 that constitute the same subsite of the ligand binding pocket. However, this interaction does not involve a conventional salt bridge, but rather an unusual lysine-aromatic interaction.

Amino Acid Sequence↗
PubMed1994

The high affinity state of the beta 2-adrenergic receptor requires unique interaction between conserved and non-conserved extracellular loop cysteines.

A disulfide bond between two extracellular cysteines, conserved in all G-protein-coupled receptors, is believed to be critical for stabilization of the ligand-binding pocket. The beta 2-adrenergic receptor (beta 2-AR) contains two conserved cysteines (Cys106 and Cys184) as well as two other extracellular cysteines (Cys190 and Cys191). The specificity of the interactions between these four cysteines has not yet been clearly established. Mutants encoding alanines for specific extracellular cysteines in the beta 2-AR gene were constructed and expressed in COS-1 and Chinese hamster ovary cells. Ala106, Ala184,190,191, and Ala106,184,190,191 mutants displayed low affinity for the beta-antagonist, 125I-cyanopindolol and insensitivity to dithiothreitol (DTT). The Ala106,191 mutant displayed an intermediate affinity and DTT sensitivity. Mutants Ala184, Ala184,190, and Ala184,191 displayed high affinity and DTT sensitivity, indicating that a solvent-accessible disulfide bond(s) is present in these mutant receptors as in the wild-type beta 2-AR. Additionally, thermal stability studies provided evidence that the extracellular disulfide bonds are essential for stabilization of the high affinity state of the receptor. These studies indicate that the covalent linkage between loops 1 and 2 of the beta 2-AR extracellular domains involves the formation of disulfide bonds, uniquely between Cys106 and Cys191, and Cys184 and Cys190, and is, thus, distinct from that of other G-protein-coupled receptors.

Amino Acid Sequence↗
PubMed1990

Serial electrophysiologic testing of drug therapy in supraventricular tachycardia related to accessory pathways.

Data are limited on the use of serial electrophysiologic testing of drug therapy in patients with supraventricular tachycardia associated with accessory pathways, including the Wolff-Parkinson-White syndrome. Twenty-four highly symptomatic patients (aged 36 +/- 11 years) with SVT related to accessory pathways underwent electrophysiologic studies to select an effective chronic oral treatment. Conventional (verapamil, propranolol, quinidine, disopyramide) and investigational (amiodarone, aprindine, propafenone) drugs were used alone and in combination if necessary. It was determined that serial electrophysiologic studies could identify potentially effective treatments in 66% of patients with reentrant SVT involving accessory pathways, and the findings were highly predictive of long-term clinical outcome.

Adult↗