Integrin proteomics

Integrin proteomics

Adhesion signalling complexes are molecularly diverse structures that associate with integrins to enable proper cell adhesion. We have developed a methodology for the affinity isolation and mass spectrometric analysis of integrin-associated complexes. Importantly, the technique isolates stabilised, ligand-engaged integrin adhesion complexes and fractionates the cell to permit the enrichment of insoluble integrin-associated cytoskeletal components. This approach has enabled us to describe the first ligand-induced integrin proteomes.

General introduction

Integrin proteomics – general intro

Integrin proteomics: building a map of adhesion molecules

Written by Adam Byron

Cells must stick to each other in the right place at the right time. This helps the body to develop and function healthily. Integrin molecules on the surface of cells are critical for normal cell adhesion. But integrins cannot do all this work alone.

Many other molecules in the cell help integrins to function properly. Some of these helper molecules decode communication signals from neighbouring cells; some let the cell respond to these signals; some provide transport within the cell, shuttling molecules that cannot get to integrins on their own.

These helper molecules all team up close to integrins in a cluster of proteins known as a protein complex. Integrin protein complexes are often called “focal adhesions” because they are focal points of cell adhesion. The collection of molecules in a protein complex is actually very … complex (excuse the pun). We still do not know what all the helper molecules are, where they cluster with integrins or when their help is needed. If we knew all this information, we would have a better understanding of how integrin signalling worked. This knowledge could lead to treatments for diseases that occur when integrins or integrin helper molecules malfunction.

Currently, it’s a bit like trying to read a map with lots of places missing. We know roughly where things are, but there could be many unknown places, and we don’t know all the roads that link the places together. Think of the places on the map as proteins in a cell and the roads as signals or interactions between the proteins. Usually, scientists start at their favourite place (protein) on the map and discover new places and roads that are nearby. Our research has developed a way to look at the whole integrin map at the same time.

Studying all of the proteins in a system is an approach known as proteomics. Our proteomics work has shown that there are hundreds of helper molecules that collect close to integrins in protein complexes. We were surprised to find that the complicated integrin map changed when the cell was in a different environment. This may help explain how communication signals from different tissues of the body make sure cells stick to each other in the right place at the right time.

How do scientists perform proteomics?

We use a technique called mass spectrometry. Find out more about mass spectrometry.

General introduction

Integrin proteomics – general intro

Integrin proteomics: building a map of adhesion molecules

Written by Adam Byron

Cells must stick to each other in the right place at the right time. This helps the body to develop and function healthily. Integrin molecules on the surface of cells are critical for normal cell adhesion. But integrins cannot do all this work alone.

Many other molecules in the cell help integrins to function properly. Some of these helper molecules decode communication signals from neighbouring cells; some let the cell respond to these signals; some provide transport within the cell, shuttling molecules that cannot get to integrins on their own.

These helper molecules all team up close to integrins in a cluster of proteins known as a protein complex. Integrin protein complexes are often called “focal adhesions” because they are focal points of cell adhesion. The collection of molecules in a protein complex is actually very … complex (excuse the pun). We still do not know what all the helper molecules are, where they cluster with integrins or when their help is needed. If we knew all this information, we would have a better understanding of how integrin signalling worked. This knowledge could lead to treatments for diseases that occur when integrins or integrin helper molecules malfunction.

Currently, it’s a bit like trying to read a map with lots of places missing. We know roughly where things are, but there could be many unknown places, and we don’t know all the roads that link the places together. Think of the places on the map as proteins in a cell and the roads as signals or interactions between the proteins. Usually, scientists start at their favourite place (protein) on the map and discover new places and roads that are nearby. Our research has developed a way to look at the whole integrin map at the same time.

Studying all of the proteins in a system is an approach known as proteomics. Our proteomics work has shown that there are hundreds of helper molecules that collect close to integrins in protein complexes. We were surprised to find that the complicated integrin map changed when the cell was in a different environment. This may help explain how communication signals from different tissues of the body make sure cells stick to each other in the right place at the right time.

How do scientists perform proteomics?

We use a technique called mass spectrometry. Find out more about mass spectrometry.

Scientific details

Integrin proteomics – scientific detail

Mass spectrometry–based proteomics

Proteomics is the study of the entire complement of proteins in a given system, such as a cell or an organism. The term “proteome” is a combination of the words “protein” and “genome”; proteomics is analogous to genomics, which is the study of an entire set of genes. Compared to genomics, proteomics is further complicated by protein modifications such as phosphorylation, ubiquitination and glycosylation, and the fact that proteins can be expressed as alternative splice forms or in distinct cell types at different times.

A number of strategies have commonly been employed to study the proteomes of particular cell types, tissues or disease states. These include two-dimensional–gel electrophoresis, antibody chips and, with increasing use recently, mass spectrometry (MS).

MS has a broad range of chemical and biological applications. In terms of its usefulness for proteomics, MS can be described as an analytical method that measures the mass-to-charge ratio of peptides generated from enzymatic digestion of proteins. From this mass-to-charge ratio data, the accurate measurement of the mass of peptides can be calculated using the charge-state of the peptide ions detected by MS. When two or more mass spectrometers are combined, a set-up commonly termed tandem-MS or MS/MS, sequence information regarding the parent peptide can be obtained. Sequence information results from the selection and fragmentation of peptides in one of the mass spectrometers, and the subsequent measurement of the mass-to-charge ratios of the peptide fragments in the other mass spectrometer. MS is often directly coupled to reverse phase liquid chromatography (LC), permitting the separation and analysis of complex mixtures of peptides, and hence proteins, as they are eluted from the LC column. A set-up such as this is called LC-MS/MS.

A number of recent technical and methodological advances have made a significant impact on the usefulness and usability of MS-based proteomics by biologists. These advances include the construction of high-resolution and sensitive mass spectrometers, the availability of complete genomic databases for a number of species and the generation of workflows for the quantification of peptides and proteins within complex mixtures. With these advances in MS, it is now not uncommon to see research articles containing quantitative data for hundreds or even thousands of proteins at a time. Thus, the global, non-candidate–based analysis of signalling events using MS is emerging as a powerful approach for investigating cellular responses to stimuli or genetic modifications.

Proteomics of cell adhesion

Adhesion to the extracellular matrix is essential for a multicellular existence. Adhesion receptors on the cell surface transduce signals that control cell morphology, movement, survival and differentiation in various developmental, homeostatic and disease processes. Integrin adhesion complexes, like other receptor-associated signalling complexes, have been refractory to proteomic analysis. This is because integrin complexes consist of a complicated mixture of transmembrane, cytoskeletal and signalling molecules, which readily fall apart when they are isolated biochemically, especially when solubilised by detergent. The complexes are also difficult to isolate because they require ligand engagement to induce macromolecular assembly. Therefore, the global analysis of adhesion complex components has been restricted to the collation and interrogation of data derived from published experimental studies. Most MS-based studies have concentrated on the analysis of post-translational modifications and binding partners of certain relatively soluble cytoplasmic adhesion proteins. An overview of some recent proteomic studies of adhesion-related biology can be found at the Cell Migration Consortium.

We have developed a methodology for the affinity isolation and mass spectrometric analysis of integrin-associated complexes. Importantly, the technique (1) isolates ligand-engaged integrin adhesion complexes, (2) stabilises the protein complexes by chemical cross-linking and (3) fractionates the cell to permit the enrichment of insoluble integrin-associated cytoskeletal components.

We have used this approach to compare the proteomes of two receptor-ligand pairs: α4β1–vascular cell adhesion molecule–1 and α5β1–fibronectin. Numerous well-characterised components of integrin adhesion complexes were detected, along with many putative novel integrin-associated proteins, which demonstrated the effectiveness of this strategy. Moreover, this approach led to the identification of regulator of chromosome condensation–2 (RCC2) as a novel regulatory component of integrin-GTPase signalling pathways, orchestrating adhesion complex assembly, cell spreading and directional cell migration. (See our Integrin proteomics gallery in Scientific image galleries for a selection of images.)

These findings represent the first experimentally defined integrin proteomes. Furthermore, the development of this workflow now allows the molecular composition of various adhesion complexes — and indeed other transmembrane receptor–ligand protein complexes — to be measured directly and presents an entry point for systems-level analyses of adhesion signalling.

Further reading

Integrin proteomics – further reading

A Byron, JD Humphries, MD Bass, D Knight and MJ Humphries (2011) Proteomic analysis of integrin adhesion complexes. Sci. Signal. 4: pt2. Full text | PubMed entry

A Byron*, MR Morgan* and MJ Humphries (2010) Adhesion signalling complexes. Curr. Biol. 20: R1063-7. Full text | PubMed entry

JD Humphries*, A Byron*, MD Bass, SE Craig, JW Pinney, D Knight and MJ Humphries (2009) Proteomic analysis of integrin-associated complexes identifies RCC2 as a dual regulator of Rac1 and Arf6. Sci. Signal. 2: ra51. Full text | PubMed entry

* These authors contributed equally to this work.

Integrin proteomics – right hand column

integrinproteomes

Related galleries

  • Integrin proteomics
  • Cell-ECM interactions gallery

See: Scientific image galleries