Oligomerization of the alpha 1a- and alpha 1b-adrenergic receptor subtypes. Potential implications in receptor internalization.
We combined biophysical, biochemical, and pharmacological approaches to investigate the ability of the alpha 1a- and alpha 1b-adrenergic receptor (AR) subtypes to form homo- and hetero-oligomers. Receptors tagged with different epitopes (hemagglutinin and Myc) or fluorescent proteins (cyan and green fluorescent proteins) were transiently expressed in HEK-293 cells either individually or in different combinations. Fluorescence resonance energy transfer measurements provided evidence that both the alpha 1a- and alpha 1b-AR can form homo-oligomers with similar transfer efficiency of approximately 0.10. Hetero-oligomers could also be observed between the alpha 1b- and the alpha 1a-AR subtypes but not between the alpha 1b-AR and the beta2-AR, the NK1 tachykinin, or the CCR5 chemokine receptors. Oligomerization of the alpha 1b-AR did not require the integrity of its C-tail, of two glycophorin motifs, or of the N-linked glycosylation sites at its N terminus. In contrast, helix I and, to a lesser extent, helix VII were found to play a role in the alpha 1b-AR homo-oligomerization. Receptor oligomerization was not influenced by the agonist epinephrine or by the inverse agonist prazosin. A constitutively active (A293E) as well as a signaling-deficient (R143E) mutant displayed oligomerization features similar to those of the wild type alpha 1b-AR. Confocal imaging revealed that oligomerization of the alpha1-AR subtypes correlated with their ability to co-internalize upon exposure to the agonist. The alpha 1a-selective agonist oxymetazoline induced the co-internalization of the alpha 1a- and alpha 1b-AR, whereas the alpha 1b-AR could not co-internalize with the NK1 tachykinin or CCR5 chemokine receptors. Oligomerization might therefore represent an additional mechanism regulating the physiological responses mediated by the alpha 1a- and alpha 1b-AR subtypes.
Stanasila
Laura
L
Institut de Pharmacologie et de Toxicologie, Université de Lausanne, 1005 Lausanne, Switzerland.
Perez
Jean-Baptiste
JB
Vogel
Horst
H
Cotecchia
Susanna
S
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D006801:Humans; D066298:In Vitro Techniques; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D018340:Receptors, Adrenergic, alpha-1 / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D018340:Receptors, Adrenergic, alpha-1; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins