Beta2-adrenergic receptor homodimers: Role of transmembrane domain 1 and helix 8 in dimerization and cell surface expression

Abstract

Even though there are hundreds of reports in the published literature supporting the hypothesis that G protein-coupled receptors (GPCR) form and function as dimers this remains a highly controversial area of research and mechanisms governing homodimer formation are poorly understood. Crystal structures revealing homodimers have been reported for many different GPCR. For adrenergic receptors, a potential dimer interface involving transmembrane domain 1 (TMD1) and helix 8 (H8) was identified in crystal structures of the beta1-adrenergic (β1-AR) and β2-AR. The purpose of this study was to investigate a potential role for TMD1 and H8 in dimerization and plasma membrane expression of functional β2-AR. Charged residues at the base of TMD1 and in the distal portion of H8 were replaced, singly and in combination, with non-polar residues or residues of opposite charge. Wild type and mutant β2-AR, tagged with YFP and expressed in HEK293 cells, were evaluated for plasma membrane expression and function. Homodimer formation was evaluated using bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and fluorescence correlation spectroscopy. Amino acid substitutions at the base of TMD1 and in the distal portion of H8 disrupted homodimer formation and caused receptors to be retained in the endoplasmic reticulum. Mutations in the proximal region of H8 did not disrupt dimerization but did interfere with plasma membrane expression. This study provides biophysical evidence linking a potential TMD1/H8 interface with ER export and the expression of functional β2-AR on the plasma membrane.

The Application of Dynamic Models to the Exploration of β1-AR Overactivation as a Cause of Heart Failure

“…Many molecular processes within a cell are carried out by molecular machines that are built from a large number of protein components organized by their PPIs (protein-protein interactions). In the experimental groups, β 1 -AR and β 2 -AR belong to the G protein-coupled receptor family that are connected through the C-terminus [ 10 ] which belongs to PPI. Although β 1 -AA does not directly bind to β 2 -AR, β 1 -AA will indirectly affect the conformation of β 2 -AR by activating β 1 -AR based on the studies of laboratory animal medicine of Capital Medical University.…”

“…u 3 represents the velocity at which undegraded B 1 ′ returns to the cell surface and participates in the reaction again. Also, β 1 -AR and β 2 -AR belong to the G protein-coupled receptor family [ 10 ], and thus we assume that they have the same degradation velocity u 1 .…”

“…A study found that, in addition to catecholamine β 1 -AR agonist ISO, β 1 -adrenoceptor ( β 1 -AA) autoantibodies that could cause continuous activation of β 1 -AR [ 9 ] were detected in 40%–60% of HF patients [ 8 ]. β 1 -AR and β 2 -AR ( β 1 -adrenergic receptors) link through the C-terminal, which are within cardiac cells, and belong to the G protein-coupled receptor family [ 10 ]. β 1 -AR and β 2 -AR can form heterodimers [ 11 ], where β 2 -AR downstream couples with stimulatory G protein ( G s ) and inhibitory G protein ( G i ) [ 12 ].…”

Visualization of class A GPCR oligomerization by image-based fluorescence fluctuation spectroscopy

“…Fluorescence fluctuation spectroscopy techniques offer an effective tool to investigate with good precision receptor dimerization at higher expression levels than SMT. Single point fluorescence correlation spectroscopy was used in the past to characterize GPCR diffusion, formation of hetero-complexes [14] and receptor homodimerization, by analyzing the histogram of the collected photons [15,16]. Time-based image fluorescence fluctuation spectroscopy methods, which rely on the statistical analysis of many pixels of an image, have allowed to characterize the oligomerization state of the GPI-anchored membrane receptor uPar [17] and the ErbB [18] observing the agonist-dependent formation of dimers and oligomers.…”

Examination of endocrine disruptor effects in neuroendocrine systems, in vivo and in vitro

“…The NE membrane receptor acts on a specific activation pathway as an agonist, via the cAMP messenger system. The transduction process starting with the NE + receptor binding from the extracellular space on the β-adrenoceptor, it highly depends on the activation (83) and integrity of the membrane (84), the membrane potential (85), the rate of movement of the receptor subunits (86) and its direction (87).…”