Corticotropin-releasing factor binding protein dimerizes after association with ligand.

Abstract

We have suggested recently that the fall in plasma CRF-binding protein (BP) during the last few weeks of pregnancy is a direct effect of association with its ligand because of the rapid decrease in plasma BP concentration seen in normal males reaching a nadir some 15 min after a bolus injection of synthetic CRF. In the present study, we have investigated the physicochemical properties of both natural and recombinant BP by gel filtration under physiological conditions and have shown that association of human CRF to this BP results in an increase in molecular weight consistent with the formation of a dimer form of the BP ligand complex. The dimer is more stable when the interaction occurs in the presence of serum or if a peptide with a higher affinity for the BP is substituted as ligand. Experimental evidence would also suggest that the dimer BP has a higher affinity for ligand than the monomeric form. We suggest that this dimerization occurs in vivo when CRF is released into the bloodstream and provides the trigger that causes the uptake of the complex at specific receptor sites.

The binding protein of corticotropin-releasing factor: Ligand-binding site and subunit structure

“…It has been proposed that CRF induces the formation of homodimers of CRFBP (15). Thus, it was conceivable that the ligand was bound to one or both subunits of this putative CRFBP complex.…”

“…4b) was assigned to rCRFBP(34-38) photolabeled by [Bp 6,32 ]h͞rCRF 6 -33 (6)(7)(8) (M calc ϭ 1,171.43; ⌬M ϭ 110 ppm). Thus, rCRFBP (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26) and rCRFBP(34-38) were identified as the binding sites of the C-and the N-terminal part of the bifunctional photoprobe, respectively. In agreement with this finding, the monofunctional photoprobes [Bp 32 ]h͞ rCRF 6 -33 and [Bp 6 ]h͞rCRF 6 -33 labeled rCRFBP (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26) and rCRFBP(34-38), respectively (data not shown).…”

“…The high-energy CID mass spectrum of the photoadduct fragment rCRFBP (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)ϫ[Bp 6,32 ]h͞rCRF 6 -33 (25)(26)(27)(28)(29)(30)(31)(32)(33) did not reveal any fragment ion signals that would be indicative of the cleavage of the peptide backbone of rCRFBP (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). Therefore, the CID mass spectra of the photoadduct fragment and of synthetic rCRFBP (12)(13)(14)(15)(16)(17)(18)(19)(...…”

“…Therefore, the CID mass spectra of the photoadduct fragment and of synthetic rCRFBP (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26) were compared with respect to their low-mass ion regions-as described above (data not shown). The lack of the low-mass ion signals at m͞z ϭ 100, 112, and 129 indicated the labeling of the side chain of Arg-23 by [Bp 6,32 ]h͞ rCRF 6 -33 (25)(26)(27)(28)(29)(30)(31)(32)(33).…”

“…New photoreactive analogs of h͞rCRF 6 -33 were used in combination with different mass spectrometric techniques to directly determine contact sites between residues of CRF and its binding protein. In view of the finding that human CRFBP dimerizes after association with ligand (15), the subunit structure of rCRFBP was investigated with mono-and bifunctional photoprobes, as well as by chemical cross-linking.…”