Reconstitution and analysis of soluble inhibin and activin receptor complexes in a cell-free system.
Activins and inhibins compose a heterogeneous subfamily within the transforming growth factor-beta (TGF-beta) superfamily of growth and differentiation factors with critical biological activities in embryos and adults. They signal through a heteromeric complex of type II, type I, and for inhibin, type III receptors. To characterize the affinity, specificity, and activity of these receptors (alone and in combination) for the inhibin/activin subfamily, we developed a cell-free assay system using soluble receptor-Fc fusion proteins. The soluble activin type II receptor (sActRII)-Fc fusion protein had a 7-fold higher affinity for activin A compared with sActRIIB-Fc, whereas both receptors had a marked preference for activin A over activin B. Although inhibin A and B binding was 20-fold lower compared with activin binding to either type II receptor alone, the mixture of either type II receptor with soluble TGF-beta type III receptor (TbetaRIII; betaglycan)-Fc reconstituted a soluble high affinity inhibin receptor. In contrast, mixing either soluble activin type II receptor with soluble activin type I receptors did not substantially enhance activin binding. Our results support a cooperative model of binding for the inhibin receptor (ActRII.sTbetaRIII complex) but not for activin receptors (type II + type I) and demonstrate that a complex composed of activin type II receptors and TbetaRIII is both necessary and sufficient for high affinity inhibin binding. This study also illustrates the utility of this cell-free system for investigating hypotheses of receptor complex mechanisms resulting from crystal structure analyses.
Program in Membrane Biology and the Renal Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
Elisabetta
E
del Re
Yisrael
Y
Sidis
David A
DA
Fabrizio
Herbert Y
HY
Lin
Alan
A
Schneyer
D029404:Activin Receptors / Q000737:chemistry*; D002460:Cell Line; D002474:Cell-Free System; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D007265:Inhibins / Q000737:chemistry*; D007455:Iodine / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011961:Receptors, Fc / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D016212:Transforming Growth Factor beta / Q000378:metabolism
D008024:Ligands; D011961:Receptors, Fc; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D007265:Inhibins; D007455:Iodine; D029404:Activin Receptors