Assays and properties of arfaptin 2 binding to Rac1 and ADP-ribosylation factors (Arfs)

Ok Ho Shin, John H. Exton

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Arfaptin 1 and 2 were identified as targets for GTP bound ADP-ribosylation factors (Arfs). Arfaptin 1 had no significant effects on guanine nucleotide binding to Arfs, nor enzymatic activities of guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP) acting on Arfs. However, arfaptin 1 inhibited Arf activation of cholera toxin and phospholipase D (PLD) in a dose-dependent manner. Only GTP-bound forms of Arf1, 5, and 6 interacted with arfaptin 1 and 2, but GTP-Arf1 showed the strongest binding to the arfaptins. In contrast to the binding of Arfs to arfaptins, GDP-Rac1 or dominant negative Rac1-N17N bound to arfaptin 2, whereas GTP-Rac1 or dominant active Rac1-Q61L did not bind to arfaptin 2. Neither GTP-Rac1 nor GDP-Rac1 bound to arfaptin 1. Based on our observation, we propose that arfaptin 2 is a target for GDP-Rac1 and for GTP-Arf1, and is involved in interactions between the Rac1 and Arfs signaling pathways. This chapter describes methods for investigating the interactions of arfaptins 1 and 2 with GTP- or GDP-liganded Arfs and Rac1.

Original languageEnglish (US)
Article number31
Pages (from-to)359-367
Number of pages9
JournalMethods in Enzymology
Volume404
DOIs
StatePublished - 2006
Externally publishedYes

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ADP-Ribosylation Factors
Guanosine Triphosphate
Assays
GTPase-Activating Proteins
Guanine Nucleotide Exchange Factors
Phospholipase D
Guanine Nucleotides
Cholera Toxin
Chemical activation
Observation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Assays and properties of arfaptin 2 binding to Rac1 and ADP-ribosylation factors (Arfs). / Shin, Ok Ho; Exton, John H.

In: Methods in Enzymology, Vol. 404, 31, 2006, p. 359-367.

Research output: Contribution to journalArticle

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