Characterization of Corticotropin-Releasing Hormone Binding Protein in Human Plasma by Chemical Cross-Linking and its Binding during Pregnancy*

Abstract

A human plasma CRH-binding protein (CRH-BP) was identified and characterized by chemical cross-linking of 125I-Tyr-hCRH to human plasma using disuccinimidyl suberate. The apparent mol wt of the cross-linked complex determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography was approximately 43,000. The mol wt was slightly lower in the nonreduced state, suggesting the presence of intramolecular disulfide bonds. Subtracting the mol wt of 125I-Tyr-CRH, the BP appeared to have a mol wt of approximately 38,000. Binding was specific since the appearance of the 43,000 dalton band was not affected by unlabeled ACTH, vasopressin, serum albumin, or gamma-globulin, but was inhibited by unlabeled hCRH dose dependently. Pretreatment of plasma with 0.1 mol/L HCl, 0.01 mol/L NaOH, 10 mmol/L dithiothreitol, or trypsin before cross-linking abolished its ability to bind 125I-Tyr-hCRH. Rat, rabbit, or goat plasma or human cerebrospinal fluid did not bind 125I-Tyr-CRH. It is unlikely that CRH-BP is a CRH receptor, because the estimated mol wt of the CRH-BP is smaller than the reported size of CRH receptors, and the CRH-BP did not bind to ovine CRH. The binding of 125I-Tyr-CRH to CRH-BP decreased in the third trimester of pregnancy, when plasma CRH levels were markedly elevated. However, after dissociating endogenous CRH from the CRH-BP, the binding was almost the same as in nonpregnant subjects. In addition, CRH-BP inhibited CRH-induced ACTH secretion from cultured rat anterior pituitary cells. We conclude that most of the increased plasma CRH found in pregnant women is bound to CRH-BP, and so is inactive, therefore plasma ACTH levels do not increase to above the normal range.

Placental corticotropin-releasing hormone may be a stimulator of maternal pituitary adrenocorticotropic hormone secretion in humans.

“…Detection of plasma ACTH in spite of the significant increase of plasma-free cortisol during pregnancy may support this hypothesis. Second, the bound CRH to CRH binding protein still has bioactivity (16). Third, maternal plasma CRH levels did not show any significant variation during a 3-h observation when plasma samples were obtained at 20-min intervals in late pregnancy (Sasaki, A., unpublished observations); in contrast, plasma CRH after a bolus injection of synthetic rat CRH was rapidly eliminated from peripheral plasma (17).…”

Residues of Corticotropin Releasing Factor-binding Protein (CRF-BP) That Selectively Abrogate Binding to CRF but Not to Urocortin 1

“…Corticotropin releasing factor-binding protein (CRF-BP) is a 37-kDa glycoprotein that was originally found to circulate in high concentrations in late gestational maternal plasma where it likely prevents inappropriate release of ACTH from the pituitary gland by placental-derived CRF (13)(14)(15). CRF-BP was named for its ability to bind to CRF with high affinity (16), but it also binds to other members of the CRF family of neuropeptides.…”

Large Molecular Weight Immunoreactive Corticotrophin‐Releasing Hormone has Bioactivity on Superfused Ovine Pituitary Cells

“…Suds et al (11) reported that the levels of CRH binding protein Data reported by other investigators on the forms of human placental CRH-IR have not been consistent, with findings of two (2, 3) and three (13) larger molecular weight forms. Sasaki et al using Sephadex G-75 chromatography, described two peaks of CRH-IR in human placental extracts (2), a result replicated by Cunnah et al (3) using similar conditions.…”