Inhibition of melanocortin-4 receptor dimerization by substitutions in intracellular loop 2

Abstract

Obesity is one of the most challenging global health problems. One key player in energy homeostasis is the melanocortin-4 receptor (MC4R), which is a family A G-protein-coupled receptor (GPCR). It has recently been shown that MC4R has the capacity to form homo-or heterodimers. Dimerization of GPCRs is of great importance for signaling regulation, with major pharmacological implications. Unfortunately, not enough is yet known about the detailed structural properties of MC4R dimers or the functional consequences of receptor dimerization. Our goal, therefore, was to explore specific properties related to MC4R dimerization. First, we aimed to induce the dissociation of dimers to monomers and to compare the functional parameters of wild-type and MC4R variants. To inhibit homodimerization, we designed MC4R chimeras with the cannabinoid-1 receptor, a receptor that does not interact with MC4R. Indeed, we identified several substitutions in the intracellular loop 2 (ICL2) and adjacent regions of transmembrane helix 3 (TMH3) and TMH4 that lead to partial dimer dissociation. Interestingly, the capacity for signaling activity was generally increased in these MC4R variants, although receptor expression remained unchanged. This increase in activity for dissociated receptors might indicate a link between receptor dimerization and signaling capacity. Moreover, dimer dissociation was also observed in a naturally occurring activating MC4R mutation in ICL2. Taken together, this study provides new information on the structural prerequisites for MC4R dimerization and identifies an approach to induce the dissociation of MC4R dimers. This might be useful for further investigation of pharmacological properties.

Oligomerization of GPCRs involved in endocrine regulation

“…(ii) Signaling of the monomer is enhanced compared with the oligomer (Piechowski et al 2013). (iii) Signaling of the monomer is weaker compared with the oligomer (Wilson et al 2007, Magalhaes et al 2010, Pellissier et al 2011).…”

“…3. A heteromer, which may also be an orphan receptor (Levoye et al 2006b), in a given cell type may: (Piechowski et al 2013). Furthermore, there may be heterooligomerization of the receptors (as described for the MC4R), indicated by receptor pairs (mixed color).…”

“…Inhibition of MC4R oligomerization could be achieved by domain substitution using the noninteracting cannabinoid-1 receptor (Piechowski et al 2013). Substitutions of the MC4R ICL2 and adjacent regions of TMH3 and TMH4, with respective regions of the cannabinoid-1 receptor, lead to partial MC4R dimer dissociation.…”

3-iodothyronamine differentially modulates α-2A-adrenergic receptor-mediated signaling

“…Receptor-receptor oligomerization was determined by a sandwich-ELISA as described previously (Piechowski et al 2013). In brief, N-terminally HA-tagged ADRA2A and C-terminally Flag-tagged TAAR1 (or vice versa) were co-expressed in COS-7 cells.…”

The Multitarget Ligand 3-Iodothyronamine Modulates β-Adrenergic Receptor 2 Signaling

“…3-T 1 AM was dissolved in DMSO and used in a concentration of 0.1% for 10 -5 M 3-T 1 AM, which did not affect cAMP accumulation. Stimulation was performed 48 h after transfection as described previously [19]. Cells were incubated for 40 min without ligand or with 3-T 1 AM (final concentration of DMSO 0.1%), ISOP or norepinephrine (NorEpi; Sigma Aldrich, St. Louis, Mo., USA; both dissolved in H 2 O), or were costimulated with ISOP, or NorEpi and 3-T 1 AM, or NorEpi with 3,3′,5-triiodothyronine (T 3 ; Sigma Aldrich; dissolved in HCl final concentration of 0.01%).…”