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Re‐Defining the Specificity of Phosphoinositide‐Binding by Human PH Domain‐Containing Proteins
Author(s) -
Singh Nilmani,
Reyes-Ordonez Adriana,
Compag Michael,
Moreno Jesus,
Yao Lucy,
Chen Jie
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.04312
Subject(s) - chemistry , total internal reflection fluorescence microscope , protein–lipid interaction , biochemistry , biophysics , plasma protein binding , fluorescence microscope , membrane protein , fluorescence , membrane , integral membrane protein , biology , physics , quantum mechanics
The human genome encodes over 250 PH domain containing proteins, all of which share an invariable structure. PH domains are presumed to mediate protein‐lipid interactions specifically to phosphoinositides (PIPs), and yet only a small fraction of PH domain‐containing proteins have been reported to bind PIPs with specificity. Most reported protein‐lipid interaction studies employ methods that either require purified proteins or utilize non‐physiological presentation of lipids, and rely on isolated PH domains rather than full‐length proteins. To circumvent these problems, we recently developed a total internal reflection fluorescence (TIRF) microscopy‐based single‐molecule pulldown (SiMPull) assay, called lipid‐SiMPull, to study protein‐lipid interactions using GFP‐tagged full‐length proteins in whole cell lysates and lipid vesicles. Here, we applied the lipid‐SiMPull assay to probe the lipid binding properties of 67 human PH domain proteins. We found that >50% of those full‐length proteins bound at least one of the phospholipids, with 44% of them displaying various levels of selectivity towards PIPs. About two‐thirds of those proteins had not been previously reported to bind PIPs with any specificity. Additionally, we examined lipid binding by 17 isolated PH domains and found that most of them did not behave like their full‐length counterparts. One of the proteins, XPLN (ARHGEF3), was found to bind specifically PI‐4,5‐P 2 in lipid‐SiMPull. Further characterization revealed that PI‐4,5‐P 2 binding was necessary for XPLN membrane association, activation of RhoA, and stimulation of stress fiber formation. Taken together, our findings suggest that specific PIP recognition by PH domain proteins is more prevalent than previously believed, and that full‐length proteins display lipid binding properties distinct from the isolated PH domains. Support or Funding Information NIH NIGMS R01GM089771