Abstract

Phosphotyrosine binding (PTB) domains are structurally conserved modules acting as adaptors or scaffolds to organize signaling complexes involved in a wide range of physiological processes. The PTB domain was first identified in the Shc signaling protein and subsequently in insulin receptor substrate 1 (IRS-1), as a modular domain that recognizes proteins with phosphorylated NPxY motifs. Structural studies have since divided phosphotyrosine-binding PTB domains into Shc-like and IRS-like based on the structure of their peptide binding grooves (see Uhlik et al 15567406). More recently, another category of PTB domains was identified as binding to non-phosphorylated (apo) NpxY/F motifs. These phosphotyrosine-independent Dab-like PTBs are thought to represent nearly 75% of proteins encoding PTB domains.

The general mode of peptide binding is conserved across all PTB domains. The PTB peptides are bound partly by beta-augmentation of several weakly conserved residues followed by the distal end containing the core consensus motif structured as a beta-turn. There are two fully conserved residues in the NxxY motif but with a strong preference for Proline at the -2 position while there is a weaker preference for a hydrophobic residue at position -5 (positions within PTB motifs are considered relative to the tyrosine residue at position 0.). The combination of both Pro -2 and hydrophobic -5 leads to higher affinity binding. The peptide backbone beta-augmentation further stabilizes the motif interactions. The core phosphorylation-independent motif is exactly the same as the aforementioned motif except with a Phe able to replace the Tyr residue (Nxx[YF]). In contrast to the pTyr motif, the apo-motif is less reliant on the Tyrosine at position 0 and instead relies on hydrophobic interactions and hydrogen bonding along the whole peptide. Furthermore, positions C-terminal of the core motif can contribute to the affinity of the binding. This is in contrast to the phosphorylated motif, that tends to branch away from the PTB surface in the residues following the pTyr.

The role of PTB domains is best characterized in the Shc adaptor protein. The presence of both PTB and SH2 domains allows Shc to bind numerous growth factors upon ligand stimulated activation. Upon binding to the activated receptor, Shc itself is phosphorylated and recruits a plethora of adaptor proteins, such as Grb2 and SOS1, leading to downstream pathway activation. To date, Shc is known to bind phosphotyrosine motifs on at least 15 different growth factors or cytokine receptors. Other PTB-containing proteins of the IRS (IRS1-4) and Dok (Dok1-5) families appear to function in a similar way (Uhlik et al. 2005 15567406).

In contrast, to the inducible nature of Shc/Irs/Dok PTB-mediated binding, almost all other PTB domains have specificities independent of phosphotyrosine. In fact, binding of many PTB domains is effectively inhibited by the presence of phosphotyrosine. Several of these Dab-like PTB containing proteins have been linked to Alzeimer's disease on the basis of their binding to amyloid precursor protein (Zhang et al. 1997 9321393). These motifs are also strongly associated with endocytic signalling, in particular, receptors containing the motif [FY].NP.[FY] are often endocytosed via adaptor proteins such as Dab1 (Bonifacino and Traub. 2003 12651740).

The ability of a motif to bind different PTB domains depending on the phosphorylation state of its tyrosine residue means the motif can act as a phosphotyrosine switch. The NPxY motif in beta-integrin tails alters in this way to regulate the binding of tensin or talin within focal adhesions (Legate and Fassler. 2009 (19118211).

It should also be noted that some, perhaps most, PTB domains have the ability to bind phospholipids. Such a dual role is also found with domains of the PDZ and PH domain families,. The simultaneous binding of phospholipids and peptide to PTB domains has been shown for non-phosphorylated motif binding Dab1-like domains (Howell et al. 1999, 10373567) whilst phosphopeptides and phospholipids have been reported to mutually compete for binding to the Shc PTB domain (Rameh et al. 1997 (9268346), Zhou et al. 1995 (8524391)). However, in the later case the recruitment of Shc by the phospholipid precedes phosphopeptide binding (Ravichandran et al. 1997 (9271429)). This ability to bind phospholipids may be crucial for the correct localization of PTB domains to the cytosolic side of cell membrane and in enabling cooperative signalling.

Recent studies on the Talin PTB domain have revealed a variant motif with low similarity to the NxxY/F motif, the SPLH motif (Kong et al (16616931)). This variant is currently not represented in ELM.