Review
Structural and Evolutionary Division of Phosphotyrosine Binding (PTB) Domains

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Proteins encoding phosphotyrosine binding (PTB) domains function as adaptors or scaffolds to organize the signaling complexes involved in wide-ranging physiological processes including neural development, immunity, tissue homeostasis and cell growth. There are more than 200 proteins in eukaryotes and nearly 60 human proteins having PTB domains. Six PTB domain encoded proteins have been found to have mutations that contribute to inherited human diseases including familial stroke, hypercholesteremia, coronary artery disease, Alzheimer's disease and diabetes, demonstrating the importance of PTB scaffold proteins in organizing critical signaling complexes. PTB domains bind both peptides and headgroups of phosphatidylinositides, utilizing two distinct binding motifs to mediate spatial organization and localization within cells. The structure of PTB domains confers specificity for binding peptides having a NPXY motif with differing requirements for phosphorylation of the tyrosine within this recognition sequence. In this review, we use structural, evolutionary and functional analysis to divide PTB domains into three groups represented by phosphotyrosine-dependent Shc-like, phosphotyrosine-dependent IRS-like and phosphotyrosine-independent Dab-like PTBs, with the Dab-like PTB domains representing nearly 75% of proteins encoding PTB domains. In addition, we further define the binding characteristics of the cognate ligands for each group of PTB domains. The signaling complexes organized by PTB domain encoded proteins are largely unknown and represents an important challenge in systems biology for the future.

Introduction

PTB-containing proteins have quickly gained notoriety as critical regulators for the spatio-temporal organization of signaling networks. The importance of PTBs is represented by the range of physiological processes they modulate, including neuronal development, immune response, tissue homeostasis, and cell growth. Indeed, the list of PTB adapter/scaffold proteins linked to human disease and pathological conditions is ever growing. Mutations in PTB domain-encoded proteins so far have been identified in five human inherited diseases including hypercholesteremia, familial stroke, coronary artery disease, Alzheimer's disease, and diabetes.1, 2, 3, 4, 5, 6, 7, 8

Since 1994, when the initial characterization of PTB domains as modular phosphotyrosine-specific peptide binding domains was made, a rather large and steady body of data concerning PTBs has been amassed. Over the past ten years, several twists on common themes have emerged that necessitates the redefining of PTB domains. Some PTB domains require phosphorylation of a tyrosine in the peptide ligand for high affinity binding, but many more PTB domains bind their target substrates independent of phosphorylated tyrosine residues. PTB domains also bind head groups of acidic phospholipids, consistent with the nearly universal subcellular localization of PTB domains to membrane or juxtamembrane regions; suggesting most PTB domains are multifunctional in their interactions with the unique ability to target both proteins and phospholipids. Historically, PTB domains were segregated into two distinct subgroups: the shorter IRS-like and the longer Shc-like PTB domains. Herein, we integrate structural, evolutionary, and functional analysis that necessitates a division of the PTB domain family into three distinct subgroups.

Section snippets

Structural features of PTB domains

A clear functional dichotomy exists within the PTB domain family that is based upon the requirement of phosphotyrosine in the peptide ligand for binding. This division places Shc/IRS/Dok/SNT PTBs in one group (pY-dependent) and all others in a separate pY-independent group (see Table 1). PTB domains have historically been categorized as “IRS-like” or “Shc-like”, because of overt structural differences.9 We have identified key structural variations in PTB domains that reveal three distinct modes

Basic anatomy of a PTB domain

Shortly, after the identification of Shc as the founding member of PTB domain proteins,10 a 3D structure of the Shc PTB domain was solved.11 The Shc PTB domain was found to adopt a structural conformation similar to that of pleckstrin homology (PH) domains. This was a bit surprising considering the low level of primary sequence conservation between these two domains. Nevertheless, each of the ten different PTB domains with structures obtained thus far share this folding pattern, commonly

Structural analysis of peptide binding

The location and spatial arrangement of the peptide binding pocket is conserved in all PTB domain structures obtained thus far.11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 Furthermore, the general mode of peptide binding within this groove is also shared (Figure 1). PTB peptide ligands are bound as an anti-parallel pseudo-β sheet forming extensive contacts with the β5 strand and the C-terminal α helix. The peptides themselves form a type I β turn towards their distal end. This kink is

Structural analysis of phospholipid binding

One common trait that all PTB domain-containing proteins appear to share is that they are localized either to juxtamembrane regions or to membranes themselves under at least a subset of conditions. This can be explained in part because most of the ligands of PTB domains are transmembrane or membrane-associated proteins, further discussed below. However, a growing number of PTB domains have been found to bind directly to liposome-associated or free phospholipid head groups. Since PTB domains

Evolutionary analysis of PTB domains

PTB domains from different classes share the general functions of peptide and phospholipid binding, but at some point must have diverged evolutionarily in their modes of peptide recognition. Using algorithms developed from structure-based sequence alignments of PTB domains it is clear that a large evolutionary divergence exists between the IRS-like and Shc/Dab-like PTBs. A search of three non-redundant protein databases (Swiss-Prot, TREMBL, and TREMBL-new) with an algorithm built upon an

Functions of PTB domains

Almost 220 distinct PTB domains have been identified in a variety of eukaryotic organisms including Caenorhabditis elegans, Anopheles gambiae, Drosophila melanogaster, Xenopus laevis, and mammals. PTB domains have not been found in Saccharomyces cerevisiae or Arabidopsis thaliana, but two PTBs do exist in Dictyostelium discoidium (Talin A and Talin B). A majority of the known PTB domains are found in mammals (around 150 total), with approximately 60 PTB-encoded proteins present in humans.

Receptor tyrosine kinase and cytokine receptor signaling

The most well characterized PTB domain-containing protein is the Shc adapter protein (reviewed by Ravichandran).48 The presence of both SH2 and PTB domains in Shc allows for its binding to a variety of growth factor receptors upon their ligand-stimulated activation. Upon binding to activated receptors, Shc itself is tyrosine phosphorylated and recruits the Grb2 adapter via Grb2 SH2-mediated binding. Sos, a Ras nucleotide exchange factor, is then bound, whereupon Ras activation stimulates the

Low density lipoprotein receptor signaling

A number of phosphotyrosine-independent PTB adapters have been shown to bind to members of the low density lipoprotein (LDL) receptor family.54 One of the first of these PTB adapters shown to bind LDL receptors was the mammalian disabled-1 (Dab-1).55, 56, 57 The function of Dab1 was initially characterized genetically using mice with the targeted disruption of the mdab1 gene58 or mice with spontaneous autosomal recessive mutations in mdab1.59 Each of these mouse lines exhibited a phenotype

Amyloid precursor protein processing

Several PTB adapter proteins have been recently linked to Alzheimer's disease on the basis of their ability to bind to amyloid precursor protein.71 Alzheimer's disease is characterized by the buildup of miliary amyloid plaques in the brain parenchyma. The major components of these amyloid plaques are the secreted Aβ peptides Aβ40 and Aβ42 derived from the transmembrane amyloid precursor protein (APP). In neuronal cells, β- and γ-secretase-mediated processing of APP occurs in endocytic vesicles.

Asymmetric cell division

Proteins of the Numb family are all involved in mediating asymmetric cell division. The C. elegans Numb homolog, CKA1, binds to the atypical protein kinase C PKC3, via its PTB domain.84, 85 PKC3 function is essential for embryogenesis and asymmetry in early cell divisions. CKA1 functions to properly sequester and localize PKC3 to lateral surfaces of polarized cells. Thus, CKA1 is presumed to be an important regulator of early development in C. elegans. Likewise, the Drosophila Numb (dNumb)

Integrin activation and cell adhesion

Integrins are cell adhesion molecules that bind to the extracellular matrix (ECM) and regulate various cellular processes including motility, polarity, growth, and survival.97 Ligand binding induces integrin clustering and establishment of focal complexes. These focal complexes are composed of adapter and signaling proteins that recruit additional factors. This leads to the development of a focal adhesion, which is a nexus for ECM–integrin–cytoskeleton signaling pathways. Many PTB domain

Future outlook

Table 3 presents a summary of the structural and functional features that define the three subgroups of PTB domains. While significant diversity exists in PTB domains, it is clear that they all function in an adapter/scaffold capacity. Six PTB domain encoded scaffold proteins have been found to have mutations that contribute to human disease (Table 4), demonstrating the importance of these proteins in organizing critical signaling complexes in cells. The signaling complexes controlled by most

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