Glycoprotein hormone receptors: link between receptor homodimerization and negative cooperativity

Abstract

The monomeric model of rhodopsin-like G protein-coupled receptors (GPCRs) has progressively yielded the floor to the concept of GPCRs being oligo(di)mers, but the functional correlates of dimerization remain unclear. In this report, dimers of glycoprotein hormone receptors were demonstrated in living cells, with a combination of biophysical (bioluminescence resonance energy transfer and homogenous time resolved fluorescence/fluorescence resonance energy transfer), functional and biochemical approaches. Thyrotropin (TSHr) and lutropin (LH/CGr) receptors form homo-and heterodimers, via interactions involving primarily their heptahelical domains. The large hormone-binding ectodomains were dispensable for dimerization but modulated protomer interaction. Dimerization was not affected by agonist binding. Observed functional complementation indicates that TSHr dimers may function as a single functional unit. Finally, heterologous bindingcompetition studies, performed with heterodimers between TSHr and LH/CG-TSHr chimeras, demonstrated the unsuspected existence of strong negative cooperativity of hormone binding. Tracer desorption experiments indicated an allosteric behavior in TSHr and, to a lesser extent, in LH/ CGr and FSHr homodimers. This study is the first report of homodimerization associated with negative cooperativity in rhodopsin-like GPCRs. As such, it may warrant revisitation of allosterism in the whole GPCR family.

Evidence for activity-regulated hormone-binding cooperativity across glycoprotein hormone receptor homomers

“…As shown previously 26,36,37 , the rate of dissociation of radiolabelled TSH from the WT receptor is strongly accelerated in the presence of a large excess of cold hormone (Fig. 1g, black symbols), and this behaviour has been shown to reflect negative cooperativity of TSHr homomers 26 .…”

“…On the basis of our assumption that negative cooperativity results from allosteric interactions between receptor protomers in a dimer or oligomer 26 , the observed reduction in allosteric behaviour of the mutants could be due to reduction of their ability to dimerize. Homogeneous time resolve fluorescence resonance energy transfer (HTRF) experiments demonstrated that constitutively active receptors are able to homomerize similarly to the WT receptor (Supplementary Fig.…”

“…1g, black symbols), and this behaviour has been shown to reflect negative cooperativity of TSHr homomers 26 . When tested under the same conditions, all mutant receptors showed a decrease in the rate of tracer dissociation compared with WT ( Fig.…”

“…When we first reported the link between receptor dimerization and negative binding cooperativity the phenomenon was observed in all three GpHrs 26 . To determine whether the relationship between negative cooperativity and hormone-independent basal activity is a general characteristic of this particular class A GPCR subfamily, we studied the allosteric behaviour of a panel of constitutively active FSHr and LH/CGr mutants (Fig.…”

“…Concerning GpHr homomers, we and others have demonstrated that negative cooperativity regulates high affinity hormone binding. Ligand binding to one of the protomers decreases the binding affinity of the other protomer, so that a single agonist molecule would bind a receptor homomer under physiological (low) hormone concentrations 25,26 . Such interprotomeric communication might allow sensitive regulation of the target cell by low hormone concentrations, while showing effective but less sensitive responses to high ligand concentrations 27 .…”