Functional rescue of beta-adrenoceptor dimerization and trafficking by pharmacological chaperones.
Site-directed mutagenesis guided by evolutionary trace analysis revealed that substitution of V179 and W183 within a cluster of evolutionarily important residues on the surface of the fourth transmembrane domain of the beta(1)-adrenergic receptor (beta(1)AR) significantly reduced the propensity of the receptor to self-assemble into homodimers as assessed by bioluminescence resonance energy transfer in living cells. These results suggest that mutation of V179 and W183 result in conformational changes that reduce homodimerization either directly by interfering with the dimerization interface or indirectly by causing local misfolding that result in reduced self-assembly. However, the mutations did not cause a general misfolding of the beta(1)AR as they did not prevent heterodimerization with the beta(2)AR. The homodimerization-compromised mutants were significantly retained in the endoplasmic reticulum (ER) and could not be properly matured and trafficked to the cell surface. Lipophilic beta-adrenergic ligands acted as pharmacological chaperones by restoring both dimerization and plasma membrane trafficking of the ER-retained dimerization-compromised beta(1)AR mutants. These results clearly indicate that homodimerization occurs early in the biosynthetic process in the ER and that pharmacological chaperones can promote both dimerization and cell surface targeting, most likely by stabilizing receptor conformations compatible with the two processes.
Kobayashi
Hiroyuki
H
Département de Biochimie, Groupe de Recherche Universitaire sur le Médicament and Institut de recherche en Immunologie et en Cancérologie, Université de Montréal, Québec, Canada.
Ogawa
Koji
K
Yao
Rong
R
Lichtarge
Olivier
O
Bouvier
Michel
M
D000318:Adrenergic beta-Agonists / Q000378:metabolism; D000319:Adrenergic beta-Antagonists / Q000378:metabolism; D000526:Alprenolol / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D020126:Brefeldin A / Q000378:metabolism; D037281:Calnexin / Q000378:metabolism; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D004252:DNA Mutational Analysis; D019281:Dimerization; D004280:Dobutamine / Q000378:metabolism; D019143:Evolution, Molecular; D006801:Humans; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011500:Protein Synthesis Inhibitors / Q000378:metabolism; D021381:Protein Transport; D018342:Receptors, Adrenergic, beta-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000318:Adrenergic beta-Agonists; D000319:Adrenergic beta-Antagonists; D018832:Molecular Chaperones; D011500:Protein Synthesis Inhibitors; D018342:Receptors, Adrenergic, beta-1; D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins; D037281:Calnexin; D020126:Brefeldin A; D004280:Dobutamine; D000526:Alprenolol; D000242:Cyclic AMP