Proteomic analysis of native metabotropic glutamate receptor 5 protein complexes reveals novel molecular constituents

• Published in: Journal of neurochemistry Vol. 91; no. 2; pp. 438 - 450
• Main Authors: Farr, Carol D., Gafken, Philip R., Norbeck, Angela D., Doneanu, Catalin E., Stapels, Martha D., Barofsky, Douglas F., Minami, Manabu, Saugstad, Julie A.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.10.2004; Blackwell
• Subjects: Algorithms; Aminoacid receptors (glycine, glutamate, gaba); Animals; Biological and medical sciences; Brain Chemistry; Cell receptors; Cell structures and functions; Chromatography, Liquid; Computational Biology; Databases, Protein; Electrophoresis; Fundamental and applied biological sciences. Psychology; Immunoblotting; Macromolecular Substances; Male; Mass Spectrometry; metabotropic glutamate receptor; Molecular and cellular biology; Precipitin Tests; Protein Binding; protein interaction; proteomics; Proteomics - methods; Rats; Rats, Sprague-Dawley; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate - chemistry; Receptors, Metabotropic Glutamate - metabolism; mglu5 glutamate receptor; protein interaction; Metabotropic receptor; metabotropic glutamate receptor; Proteomics; Molecular interaction; Mass spectrometry; Protein
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1111/j.1471-4159.2004.02735.x

We used a proteomic approach to identify novel proteins that may regulate metabotropic glutamate receptor 5 (mGluR5) responses by direct or indirect protein interactions. This approach does not rely on the heterologous expression of proteins and offers the advantage of identifying protein interactions in a native environment. The mGluR5 protein was immunoprecipitated from rat brain lysates; co‐immunoprecipitating proteins were analyzed by mass spectrometry and identified peptides were matched to protein databases to determine the correlating parent proteins. This proteomic approach revealed the interaction of mGluR5 with known regulatory proteins, as well as novel proteins that reflect previously unidentified molecular constituents of the mGluR5‐signaling complex. Immunoblot analysis confirmed the interaction of high confidence proteins, such as phosphofurin acidic cluster sorting protein 1, microtubule‐associated protein 2a and dynamin 1, as mGluR5‐interacting proteins. These studies show that a proteomic approach can be used to identify candidate interacting proteins. This approach may be particularly useful for neurobiology applications where distinct protein interactions within a signaling complex can dramatically alter the outcome of the response to neurotransmitter release, or the disruption of normal protein interactions can lead to severe neurological and psychiatric disorders.