While it is well established that a protein's three-dimensional structure determines its function, a large fraction of proteins and protein regions lack stable structure. Such intrinsically disordered proteins contain extended regions that do not fold into a native fixed conformation 
. These disordered regions are widespread across the tree of life, particularly in eukaryotes 
. For example, amino acids comprising approximately 30–40% of the human proteome are predicted to reside within disordered regions 
. Many different functions have been ascribed to disordered proteins. For instance, they have been shown to carry out regulatory functions associated with signal transduction and molecular recognition, including transcription, protein phosphorylation, mRNA metabolism, RNA processing, translation, chaperone activity and regulation of the cell cycle 
Alternative splicing (AS) and post-translational modification such as phosphorylation are known to regulate and diversify the functions of proteins and are thought to partly account for the increased complexity of metazoan species. Human alternatively spliced exons are enriched in regions of intrinsic disorder, presumably to provide functional and regulatory diversity while avoiding disruption to core protein structure 
. Moreover, we and others have recently shown that tissue-regulated alternative exons are enriched in highly disordered regions of proteins where they frequently modulate interactions in protein-protein interaction networks 
. In addition, disordered regions often harbor linear motifs that mediate recognition functions and therefore can be considered as a class of functional domain 
Finally, intrinsic disorder is abundant among proteins associated with various human diseases such as cancer, cardiovascular disease, amyloidoses, diabetes, neurodegenerative diseases and others 
. Furthermore, highly connected proteins in “diseasome” networks are enriched in disorder 
. However, due to the wide range of roles of disordered proteins it has been difficult to ascribe specific functions to disordered regions.
In order to better understand the roles of intrinsic disorder, we previously developed a method to analyze the conservation of intrinsic disorder across the yeast clade 
. Over large regions of proteins, the property of disorder is highly conserved, i.e., the same residues are disordered in most orthologous proteins. Additionally, the underlying amino acid sequence of the disordered regions may either be conserved or significantly diverged. Based on this observation, we defined two types of conserved disorder: 1) “constrained disorder”, regions where the amino acid sequence is well conserved, and 2) “flexible disorder”, regions where the amino acid sequence has diverged. Our analyses revealed that these two types of conserved disorder have different biophysical and biological properties. Flexible disorder is predominantly associated with signaling and regulation, whereas constrained disorder is associated with chaperones and ribosomal proteins.
Here, we investigate the roles of these different forms of disorder in metazoans, with a focus on the human proteome. We provide evidence for distinct roles for disorder in tissue-specific regulation. In particular, we find different roles for constrained and flexible disorder in relation to alternatively spliced regions of proteins, phosphorylation sites and short linear motifs. While flexible disorder may predominantly function by providing structural flexibility that enables the expression and folding of splice isoforms, constrained disorder appears to provide structural scaffolding for presentation of linear motifs and phosphorylation sites, enabling tissue-regulated alternative splicing to rewire signaling pathways and protein interaction networks.