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.
del Re
Elisabetta
E
Program in Membrane Biology and the Renal Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
Sidis
Yisrael
Y
Fabrizio
David A
DA
Lin
Herbert Y
HY
Schneyer
Alan
A
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