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+.
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