Oligomerization of the α1a- and α1b-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 similar to0.10. 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 beta(2)-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 alpha(1)-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.