Journal
BIOCHEMISTRY
Volume 40, Issue 29, Pages 8572-8580Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi0103516
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- NHLBI NIH HHS [R01-HL64713] Funding Source: Medline
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PDZ domains bind to the carboxyl-termini of target proteins, and some PDZ domains are capable of oligomerization to facilitate the formation of intracellular signaling complexes. The Na+/H+ exchanger regulatory factor (NHERF-1; also called EBP50) and its relative NHERF-2 (also called E3KARP, SIP-1, and TKA-1) both have two PDZ domains. We report here that the PDZ domains of NHERF-1 and NHERF-2 bind specifically to each other but not to other PDZ domains. Purified NHERF-2 PDZ domains associate with each other robustly in the absence of any associated proteins, but purified NHERF-1 PDZ domains associate with each other only weakly when examined alone. The oligomerization of the NHERF-1 PDZ domains is greatly facilitated when they are bound with carboxyl-terminal ligands, such as the carboxyl-termini of the beta (2)-adrenergic receptor or the platelet-derived growth factor receptor. Oligomerization of full-length NHERF-1 is also enhanced by mutation of serine 289 to aspartate (S289D), which mimics the phosphorylated form of NHERF-1. Co-immunoprecipitation experiments with differentially tagged versions of the NHERF proteins reveal that NHERF-1 and NHERF-2 form homo- and hetero-oligomers in a cellular context. A point-mutated version of NHERF-1 (S289A), which cannot be phosphorylated on serine 289, exhibits a reduced capacity for co-immunoprecipitation from cells. These studies reveal that both NHERF-1 and NHERF-2 can oligomerize, which may facilitate NHERF-mediated formation of cellular signaling complexes. These studies furthermore reveal that oligomerization of NHERF-1, but not NHERF-2, is highly regulated by association with other proteins and by phosphorylation.
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