A highly conserved tyrosine residue in G protein-coupled receptors is required for agonist-mediated beta 2-adrenergic receptor sequestration.
An aromatic residue, tyrosine 326 in the prototypical human beta 2-adrenergic receptor, exists in a highly conserved sequence motif in virtually all members of the G protein-coupled receptor family. The potential role of this conserved aromatic amino acid residue in the cellular processes of sequestration (a rapid internalization of the surface receptor) and down-regulation (a slower loss of total cellular receptors) associated with agonist-mediated desensitization of the beta 2-adrenergic receptor was assessed by replacing tyrosine residue 326 with an alanine residue (beta 2AR-Y326A). This mutation completely abolishes agonist-mediated receptor sequestration without affecting the ability of the receptor to activate maximally adenylyl cyclase, to undergo rapid desensitization, and to down-regulate in response to agonist. The only other major change associated with the mutated receptor is a complete loss of the ability to resensitize following rapid desensitization. These results imply that this tyrosine residue, which is part of a highly conserved sequence motif in G protein-coupled receptors, may be responsible for their agonist-mediated sequestration and that sequestration and down-regulation of the receptor are dissociable phenomena. The lack of resensitization in the sequestration-defective beta 2-adrenergic receptor mutant strongly suggests that the sequestration pathway is an important mechanism by which cells re-establish the normal responsiveness of G protein-coupled receptors following the removal of agonist.
Barak
L S
LS
Howard Hughes Medical Institute Laboratories, Duke University Medical Center, Durham, North Carolina 27710.
Tiberi
M
M
Freedman
N J
NJ
Kwatra
M M
MM
Lefkowitz
R J
RJ
Caron
M G
MG
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D017124:Conserved Sequence; D006224:Cricetinae; D015536:Down-Regulation; D000939:Epitopes / Q000032:analysis; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D020120:Iodocyanopindolol; D007545:Isoproterenol / Q000494:pharmacology*; D007700:Kinetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D010869:Pindolol / Q000031:analogs & derivatives / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011869:Radioligand Assay; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014443:Tyrosine
D000939:Epitopes; D018343:Receptors, Adrenergic, beta-2; D011956:Receptors, Cell Surface; D014443:Tyrosine; D020120:Iodocyanopindolol; D010869:Pindolol; D019204:GTP-Binding Proteins; D007545:Isoproterenol; D000409:Alanine