Structural Studies on the PH Domains of Dbl, Sosl, IRS-1, and βARK1 and Their Differential Binding to Gβγ Subunits

Daruka Mahadevan, Narmada Thanki, Juswinder Singh, Peter McPhie, Daniela Zangrilli, Ling Mei Wang, Carmen Guerrero, Harry LeVine, Christine Humblet, Jose Saldanha, J. Silvio Gutkind, Taraneh Najmabadi-Haske

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62 Scopus citations

Abstract

Pleckstrin homology (PH) domains are ~110 amino acid residues in length and are structurally conserved in a number of intracellular signaling proteins. A role for these domains has been postulated for βARK, which binds to Gβγ subunits. We have quantified the binding of individual (His)6-tag PH domains of human Dbl, human Sosl, rat IRS-1, human βARK, and human βARK with an extra 33- residue C-terminal extension (βARK+C) to Gβγ subunits. Our in vitro binding studies show that all of the PH domains (apart from Sosl), bind Gβγ subunits in a dose-dependent manner, but βARK+C binds 4 times as much Gβγ at saturation as the others. The IRS-1 PH domain has a similar half-maximal concentration of Gβγ binding (18 nM) to βARK+C (30 nM), suggesting that the IRS-1 PH domain has sufficient determinants for Gpy binding. The βARK PH domain alone has a half-maximal value of 45 nM but a drastically reduced extent of Gβγ binding, suggesting that both the PH domain and the C-terminal 33 residues are necessary for maximal binding. Dbl has a half-maximum concentration of GpY binding of 45 nM and a maximal extent of binding similar to that of βARK, but it is difficult to demonstrate saturable binding of Gβγ to Sosl. Since it was previously predicted that the C-terminal PH domain of Pleckstrin [Tyers, M., et al. (1988) Nature 333, 470-473] contains a potential calcium binding site, we have tested the different PH domains for calcium binding. Only the PH domain of Dbl bound 45Ca2+ with a Kd of 10µM. CD spectroscopy of the purified recombinant PH domains indicated that they are predominantly β-sheet structures. Furthermore, the CD spectrum of the Dbl PH domain was significantly altered in the presence of 10µM Ca2+.

Original languageEnglish (US)
Pages (from-to)9111-9117
Number of pages7
JournalBiochemistry
Volume34
Issue number28
DOIs
StatePublished - Jul 1995
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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    Mahadevan, D., Thanki, N., Singh, J., McPhie, P., Zangrilli, D., Wang, L. M., Guerrero, C., LeVine, H., Humblet, C., Saldanha, J., Silvio Gutkind, J., & Najmabadi-Haske, T. (1995). Structural Studies on the PH Domains of Dbl, Sosl, IRS-1, and βARK1 and Their Differential Binding to Gβγ Subunits. Biochemistry, 34(28), 9111-9117. https://doi.org/10.1021/bi00028a021