Roles of threonine 192 and asparagine 382 in agonist and antagonist interactions with M1 muscarinic receptors.
Conserved amino acids, such as Thr in transmembrane domains (TM) V and Asn in TM VI of muscarinic receptors, may be important in agonist binding and/or receptor activation. In order to determine the functional roles of Thr192 and Asn382 in human M1 receptors in ligand binding and receptor activation processes, we created and characterized mutant receptors with Thr192 or Asn382 substituted by Ala. HM1 wild-type (WT) and mutant receptors [HM1(Thr192Ala) and HM1(Asn382Ala)] were stably expressed in A9 L cells. The Kd values for 3H-(R)-QNB and Ki values for other classical muscarinic antagonists were similar at HM1(WT) and HM1(Thr192Ala) mutant receptors, yet higher at HM1(Asn382Ala) mutant receptors. Carbachol exhibited lower potency and efficacy in stimulating PI hydrolysis via HM1(Thr192Ala) mutant receptors, and intermediate agonist activity at the HM1(Asn382Ala) mutant receptors. The Asn382 residue in TM VI but not the Thr192 residue in TM V of the human M1 receptor appears to participate directly in antagonist binding. Both Thr192 and Asn382 residues are involved differentially in agonist binding and/or receptor activation processes, yet the Asn382 residue is less important than Thr192 in agonist activation of M1 receptors. Molecular modelling studies indicate that substitution of Thr192 or Asn382 results in the loss of hydrogen-bond interactions and changes in the agonist binding mode associated with an increase in hydrophobic interactions between ligand and receptor.
Huang
X P
XP
Center for Drug Design and Development, Department of Medicinal & Biological Chemistry, College of Pharmacy, The University of Toledo, Ohio 43606-3390, USA.
Nagy
P I
PI
Williams
F E
FE
Peseckis
S M
SM
Messer
W S
WS
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001216:Asparagine / Q000737:chemistry / Q000502:physiology*; D001665:Binding Sites; D001667:Binding, Competitive; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D006801:Humans; D008958:Models, Molecular; D018721:Muscarinic Agonists / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D018727:Muscarinic Antagonists / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D010716:Phosphatidylinositols / Q000378:metabolism; D010890:Pirenzepine / Q000494:pharmacology; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D011869:Radioligand Assay; D043643:Receptor, Muscarinic M1; D011976:Receptors, Muscarinic / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D012601:Scopolamine / Q000494:pharmacology; D013912:Threonine / Q000737:chemistry / Q000502:physiology*; D014316:Tritium
D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D010716:Phosphatidylinositols; D043643:Receptor, Muscarinic M1; D011976:Receptors, Muscarinic; D014316:Tritium; D013912:Threonine; D010890:Pirenzepine; D011813:Quinuclidinyl Benzilate; D001216:Asparagine; D012601:Scopolamine