Assembly of TbetaRI:TbetaRII:TGFbeta ternary complex in vitro with receptor extracellular domains is cooperative and isoform-dependent.
Transforming growth factor-beta (TGFbeta) isoforms initiate signaling by assembling a heterotetrameric complex of paired type I (TbetaRI) and type II (TbetaRII) receptors on the cell surface. Because two of the ligand isoforms (TGFbetas 1, 3) must first bind TbetaRII to recruit TbetaRI into the complex, and a third (TGFbeta2) requires a co-receptor, assembly is known to be sequential, cooperative and isoform-dependent. However the source of the cooperativity leading to recruitment of TbetaRI and the universality of the assembly mechanism with respect to isoforms remain unclear. Here, we show that the extracellular domain of TbetaRI (TbetaRI-ED) binds in vitro with high affinity to complexes of the extracellular domain of TbetaRII (TbetaRII-ED) and TGFbetas 1 or 3, but not to either ligand or receptor alone. Thus, recruitment of TbetaRI requires combined interactions with TbetaRII-ED and ligand, but not membrane attachment of the receptors. Cell-based assays show that TbetaRI-ED, like TbetaRII-ED, acts as an antagonist of TGFbeta signaling, indicating that receptor-receptor interaction is sufficient to compete against endogenous, membrane-localized receptors. On the other hand, neither TbetaRII-ED, nor TbetaRII-ED and TbetaRI-ED combined, form a complex with TGFbeta2, showing that receptor-receptor interaction is insufficient to compensate for weak ligand-receptor interaction. However, TbetaRII-ED does bind with high affinity to TGFbeta2-TM, a TGFbeta2 variant substituted at three positions to mimic TGFbetas 1 and 3 at the TbetaRII binding interface. This proves both necessary and sufficient for recruitment of TbetaRI-ED, suggesting that the three different TGFbeta isoforms induce assembly of the heterotetrameric receptor complex in the same general manner.
Zúñiga
Jorge E
JE
Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, USA.
Groppe
Jay C
JC
Cui
Yumin
Y
Hinck
Cynthia S
CS
Contreras-Shannon
Verónica
V
Pakhomova
Olga N
ON
Yang
Junhua
J
Tang
Yuping
Y
Mendoza
Valentín
V
López-Casillas
Fernando
F
Sun
LuZhe
L
Hinck
Andrew P
AP
D030201:Activin Receptors, Type I / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D017930:Genes, Reporter; D014644:Genetic Variation; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular; D010766:Phosphorylation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D051899:Smad2 Protein / Q000032:analysis / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
D008024:Ligands; D020033:Protein Isoforms; D018125:Receptors, Transforming Growth Factor beta; D011994:Recombinant Proteins; C497782:SMAD2 protein, human; D051899:Smad2 Protein; D016212:Transforming Growth Factor beta; D008156:Luciferases; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II