Conformational change or proteolysis of LTBP; release of mature TGFp

Mature form

(homo- or hetero-dimer)

disulfide linkages (Figure 14-1b). These form a structure, called a cystine knot, that is relatively resistant to denatura-tion. An additional N-terminal cysteine in each monomer links TGFp monomers into functional homodimers and het-erodimers. Much of the sequence variation among different TGFp proteins is observed in the N-terminal regions, the loops joining the p strands, and the a helices. Different het-erodimeric combinations may increase the functional diversity of these proteins beyond that generated by differences in the primary sequence of the monomer.

M FIGURE 14-1 Formation and structure of TGFp superfamily of signaling molecules. (a) TGFp precursors are cleaved soon after being secreted. The pro-domain and mature domain are stored in the extracellular matrix in a complex that also contains latent TGFp-binding protein (LTBP). The mature domain contains six conserved cysteine residues (yellow circles), which form three intrachain disulfide bonds and also a single disulfide bond connecting two monomers. Following proteolysis or a conformational change in LTBP the active homo- or heterodimeric protein is released. (b) In this ribbon diagram of mature TGFp dimer, the two subunits are shown in green and blue. Disulfide-linked cysteine residues are shown in ball-and-stick form. The three intrachain disulfide linkages (red) in each monomer form a cystine-knot domain, which is resistant to degradation. [Part (a) see J. Massague and Y-G. Chen, 2000, Genes and Devel. 14:627; part (b) from S. Daopin et al., 1992, Science 257:369.]

TGFP Signaling Receptors Have Serine/Threonine Kinase Activity

To identify the cell-surface TGFp receptors, investigators first reacted the purified growth factor with the radioisotope iodine-125 (125I) under conditions such that the radioisotope covalently binds to exposed tyrosine residues. The 125I-labeled TGFp protein was incubated with cultured cells, and the incubation mixture then was treated with a chemical agent that covalently cross-linked the labeled TGFp to its receptors on the cell surface. Purification of the labeled receptors revealed three different polypeptides with apparent molecular weights of 55, 85, and 280 kDa, referred to as types RI, RII, and RIII TGFp receptors, respectively.

The most abundant TGFp receptor, RIII, is a cell-surface proteoglycan, also called ft-glycan, which binds and concentrates TGFp near the cell surface. The type I and type II receptors are dimeric transmembrane proteins with serine/ threonine kinases as part of their cytosolic domains. RII is a constitutively active kinase that phosphorylates itself in the absence of TGFp. Binding of TGFp induces the formation of complexes containing two copies each of RI and RII. An RII subunit then phosphorylates serine and threonine residues in a highly conserved sequence of the RI subunit adjacent to the cytosolic face of the plasma membrane, thereby activating the RI kinase activity.

Activated Type I TGFP Receptors Phosphorylate Smad Transcription Factors

Researchers identified the transcription factors downstream from TGFp receptors in Drosophila from genetic studies similar to those used to dissect receptor tyrosine kinase pathways (see Section 14.3). These transcription factors in Drosophila and the related vertebrate proteins are now called Smads. Three types of Smad proteins function in the TGFp signaling pathway: receptor-regulated Smads (R-Smads), co-Smads, and inhibitory or antagonistic Smads (I-Smads).

As depicted in Figure 14-2, R-Smads contain two domains, MH1 and MH2, separated by a flexible linker region. The N-terminal MH1 domain contains the specific DNA-binding segment and also a sequence called the nuclear-localization signal (NLS) that is required for protein transport into the nucleus (Chapter 12). When R-Smads are in their inactive, nonphosphorylated state, the NLS is masked and the MH1 and MH2 domains associate in such a way that they cannot bind to DNA or to a co-Smad. Phos-phorylation of three serine residues near the C-terminus of an R-Smad (Smad2 or Smad3) by activated type I TGFp re


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