Biochemical Properties of Synaptic Proteins Are Dependent on Tissue Preparation: NMDA Receptor Solubility Is Regulated by the C-Terminal Tail.

Autor: Won S; Receptor Biology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA., Sweeney CL; Genetic Immunotherapy Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA., Roche KW; Receptor Biology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA.
Jazyk: angličtina
Zdroj: Journal of cellular biochemistry [J Cell Biochem] 2024 Oct 06, pp. e30664. Date of Electronic Publication: 2024 Oct 06.
DOI: 10.1002/jcb.30664
Abstrakt: Synaptic proteins are essential for neuronal development, synaptic transmission, and synaptic plasticity. The postsynaptic density (PSD) is a membrane-associated structure at excitatory synapses, which is composed of a huge protein complex. To understand the interactions and functions of PSD proteins, researchers have employed a variety of imaging and biochemical approaches including sophisticated mass spectrometry. However, the field is lacking a systematic comparison of different experimental conditions and how they might influence the study of the PSD interactome isolated from various tissue preparations. To evaluate the efficiency of several common solubilization conditions, we isolated receptors, scaffolding proteins, and adhesion molecules from brain tissue or primary cultured neurons or human forebrain neurons differentiated from induced pluripotent stem cells (iPSCs). We observed some striking differences in solubility. We found that N-methyl-d-aspartate receptors (NMDARs) and PSD-95 are relatively insoluble in brain tissue, cultured neurons, and human forebrain neurons compared to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptors (AMPARs) or SAP102. In general, synaptic proteins were more soluble in primary neuronal cultures and human forebrain neurons compared to brain tissue. Interestingly, NMDARs are relatively insoluble in HEK293T cells suggesting that insolubility does not directly represent the synaptic fraction but rather it is related to a detergent-insoluble fraction such as lipid rafts. Surprisingly, truncation of the intracellular carboxyl-terminal tail (C-tail) of NMDAR subunits increased NMDAR solubility in HEK293T cells. Our findings show that detergent, pH, and temperature are important for protein preparations to study PSD protein complexes, and NMDAR solubility is regulated by its C-tail, thus providing a technical guide to study synaptic interactomes and subcellular localization of synaptic proteins.
(© Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Journal of Cellular Biochemistry published by Wiley Periodicals LLC.)
Databáze: MEDLINE
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