A Soluble Form of the First Extracellular Domain of Mouse Type 2β Corticotropin-releasing Factor Receptor Reveals Differential Ligand Specificity

Marilyn H Perrin,Michael R Digruccio,Steven C Koerber,Jean E Rivier,Koichi S Kunitake,Deborah L Bain,Wolfgang H Fischer,Wylie W Vale

doi:10.1074/jbc.m210476200

Marilyn H Perrin, Michael R Digruccio + Show 6 more

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https://doi.org/10.1074/jbc.m210476200

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Abstract

The heptahelical receptors for corticotropin-releasing factor (CRF), CRFR1 and CRFR2, display different specificities for CRF family ligands: CRF and urocortin I bind to CRFR1 with high affinity, whereas urocortin II and III bind to this receptor with very low affinities. In contrast, all the urocortins bind with high affinities, and CRF binds with lower affinity to CRFR2. The first extracellular domain (ECD1) of CRFR1 is important for ligand recognition. Here, we characterize a bacterially expressed soluble protein, ECD1-CRFR2beta, corresponding to the ECD1 of mouse CRFR2beta. The K(i) values for binding to ECD1-CRFR2beta are: astressin = 10.7 (5.4-21.1) nm, urocortin I = 6.4 (4.7-8.7) nm, urocortin II = 6.9 (5.8-8.3) nm, CRF = 97 (22-430) nm, urocortin III = sauvagine >200 nm. These affinities are similar to those for binding to a chimeric receptor in which the ECD1 of CRFR2beta replaces the ECD of the type 1B activin receptor (ALK4). The ECD1-CRFR2beta possesses a disulfide arrangement identical to that of the ECD1 of CRFR1, namely Cys(45)-Cys(70), Cys(60)-Cys(103), and Cys(84)-Cys(118). As determined by circular dichroism, ECD1-CRFR2beta undergoes conformational changes upon binding astressin. These data reinforce the importance of the ECD1 of CRF receptors for ligand recognition and raise the interesting possibility that different ligands having similar affinity for the full-length receptor may, nevertheless, have different affinities for microdomains of the receptor.

Highlights

Corticotropin-releasing factor (CRF)1 [1] is the major neuroregulator of the hypothalamic-pituitary-adrenal axis and, among other roles, serves to integrate the endocrine, autonomic, and behavioral responses to stress
We find that the relative affinities of the corticotropin-releasing factor (CRF) ligands for the soluble protein are similar to those for a chimeric receptor in which the ECD1 of CRFR2␤ is anchored in the plasma membrane by a single transmembrane domain of the type 1 activin receptor [32]
We found that the amino acids corresponding to the putative signal peptide of the ECD1 were cleaved in the mature CRFR1, expressed in mammalian cells

Summary

Introduction

Corticotropin-releasing factor (CRF)1 [1] is the major neuroregulator of the hypothalamic-pituitary-adrenal axis and, among other roles, serves to integrate the endocrine, autonomic, and behavioral responses to stress. In addition to its central nervous system actions, CRF and its related ligands affect the cardiovascular, reproductive, gastrointestinal, skin, and immune systems [2]. CRF receptors have been characterized in the central nervous system and various peripheral sites including pituitary, gastrointestinal tract, epididymis, heart, gonad, adrenal, skin, and skeletal muscle. The CRF receptors are 7-transmembrane domain proteins with relatively large first extracellular domains (ECD1s). Both CRFR1 and CRFR2 exist as multiple splice variants and belong to the type B receptor family that includes receptors for growth hormone-releasing factor, secretin, calcitonin, vasoactive intestinal peptide, glucagon, glucagon-like peptide (GLP), and parathyroid hormone [2]

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