Glycoprotein hormone receptors: link between receptor homodimerization and negative cooperativity.
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 binding-competition 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.
Urizar
Eneko
E
IRIBHM, Université Libre de Bruxelles, Campus Erasme, Brussels, Belgium.
Montanelli
Lucia
L
Loy
Tiffany
T
Bonomi
Marco
M
Swillens
Stéphane
S
Gales
Céline
C
Bouvier
Michel
M
Smits
Guillaume
G
Vassart
Gilbert
G
Costagliola
Sabine
S
D000494:Allosteric Regulation; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000096:biosynthesis; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D006736:Horses; D006801:Humans; D007668:Kidney; D008164:Luminescent Proteins / Q000032:analysis; D008958:Models, Molecular; D011485:Protein Binding; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011962:Receptors, FSH / Q000737:chemistry* / Q000378:metabolism; D011974:Receptors, LH / Q000737:chemistry* / Q000378:metabolism; D011989:Receptors, Thyrotropin / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D034421:Sus scrofa; D013961:Thyroid Gland / Q000378:metabolism; D014162:Transfection
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011962:Receptors, FSH; D011974:Receptors, LH; D011989:Receptors, Thyrotropin; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D000242:Cyclic AMP