TY - JOUR
T1 - Agonist binding and activation of the rat β1-adrenergic receptor
T2 - Role of Trp134 (3.28), Ser190 (4.57) and Tyr356 (7.43)
AU - Rezmann-Vitti, Linda A.
AU - Louis, Simon N.S.
AU - Nero, Tracy L.
AU - Jackman, Graham P.
AU - Iakovidis, Dimitri
AU - Machida, Curtis A.
AU - Louis, William J.
N1 - Funding Information:
We thank Mr. David Casley for iodinating cyanopindolol, Ms. Heddy Wilshire for excellent technical assistance and Dr. Ian Gordon from the Statistical Consulting Centre at The University of Melbourne for assistance with the statistical analysis. This work was supported through grants from the Austin Hospital Medical Research Foundation and the C. Edward Dunlop Foundation. Simon N.S. Louis is supported by a National Health and Medical Research Council of Australia, INSERM Fellowship.
PY - 2004/8/15
Y1 - 2004/8/15
N2 - We investigated the role of Trp134 (3.28), Ser 190 (4.57) and Tyr356 (7.43) in agonist binding to, and activation of, the rat β1-adrenergic receptor by comparing pKis and functional responses of W134A, S190A and Y356F mutant receptors to wild type, all stably expressed in CHO cells. All three mutations significantly (P < 0.05) reduced adenylyl cyclase intrinsic activity (IA) compared to wild type in response to stimulation with both (-)-isoprenaline (53-88%) and (-)-RO363 (46-61%), and there was no significant correlation either between IA or pD2 and pKi (P > 0.4), suggesting that changes in pKi were not sufficient to explain the fall in adenylyl cyclase activity. The most pronounced reduction in affinity (126-fold, P < 0.01) was displayed by xamoterol for the Y356F mutation, suggesting that xamoterol is able to directly interact with Tyr356 (7.43). For the other agonists, the change in pKi values for the mutant receptors ranged from a 20-fold decrease to a 2-fold increase compared to the wild type. In a three-dimensional model of the rat β1-adrenergic receptor, Trp134 (3.28) and Tyr356 (7.43) form part of a hydrophobic binding pocket involving residues in transmembrane helices 1, 2, 3 and 7. Our results suggest that Trp134 (3.28) and Tyr356 (7.43), together with Trp353 (7.40), are able to interact via π-π interactions to stabilize the extracellular ends of transmembrane helices 3 and 7. Ser190 (4.57) appears to be involved in a hydrogen bonding network, which maintains the spatial relationship between transmembrane helices 3 and 4. These interhelical interactions suggest that the three mutated residues stabilize the active receptor state by maintaining the proper packing of their respective transmembrane helix within the helix bundle, facilitating the appropriate movement and rotation of the transmembrane regions during the activation process.
AB - We investigated the role of Trp134 (3.28), Ser 190 (4.57) and Tyr356 (7.43) in agonist binding to, and activation of, the rat β1-adrenergic receptor by comparing pKis and functional responses of W134A, S190A and Y356F mutant receptors to wild type, all stably expressed in CHO cells. All three mutations significantly (P < 0.05) reduced adenylyl cyclase intrinsic activity (IA) compared to wild type in response to stimulation with both (-)-isoprenaline (53-88%) and (-)-RO363 (46-61%), and there was no significant correlation either between IA or pD2 and pKi (P > 0.4), suggesting that changes in pKi were not sufficient to explain the fall in adenylyl cyclase activity. The most pronounced reduction in affinity (126-fold, P < 0.01) was displayed by xamoterol for the Y356F mutation, suggesting that xamoterol is able to directly interact with Tyr356 (7.43). For the other agonists, the change in pKi values for the mutant receptors ranged from a 20-fold decrease to a 2-fold increase compared to the wild type. In a three-dimensional model of the rat β1-adrenergic receptor, Trp134 (3.28) and Tyr356 (7.43) form part of a hydrophobic binding pocket involving residues in transmembrane helices 1, 2, 3 and 7. Our results suggest that Trp134 (3.28) and Tyr356 (7.43), together with Trp353 (7.40), are able to interact via π-π interactions to stabilize the extracellular ends of transmembrane helices 3 and 7. Ser190 (4.57) appears to be involved in a hydrogen bonding network, which maintains the spatial relationship between transmembrane helices 3 and 4. These interhelical interactions suggest that the three mutated residues stabilize the active receptor state by maintaining the proper packing of their respective transmembrane helix within the helix bundle, facilitating the appropriate movement and rotation of the transmembrane regions during the activation process.
KW - CHO
KW - Chinese hamster ovary
KW - G-protein-coupled receptor
KW - GPCR
KW - M
KW - M muscarinic acetylcholine receptor
KW - WT
KW - adenosine 3′:5′-cyclic monophosphate
KW - cAMP
KW - tm
KW - transmembrane
KW - wild type
KW - β-AR
KW - β-adrenergic receptor
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U2 - 10.1016/j.bcp.2004.04.021
DO - 10.1016/j.bcp.2004.04.021
M3 - Article
C2 - 15276075
AN - SCOPUS:3242681532
SN - 0006-2952
VL - 68
SP - 675
EP - 688
JO - Biochemical Pharmacology
JF - Biochemical Pharmacology
IS - 4
ER -