A systematic mutational analysis of hormone-binding determinants in the human growth hormone receptor.

Abstract

A mutational strategy is presented that allowed us to identify hormone-binding determinants in the extracellular portion of the human growth hormone receptor (hGHbp), a 238-residue protein with sequence homology to a number of cytokine receptors. By systematically replacing side chains with alanine we probed the importance of charged residues (49 total, typically located on the surface), aromatic residues (9 total), and neighbors of these (26 total). The alanine substitutions that were most disruptive to hormone binding are located predominantly in four segments of a cysteine-rich domain in the hGHbp, and collectively they form a patch when mapped upon a structural model proposed for cytokine receptors. Control experiments with monoclonal antibodies confirmed that most of these alanine substitutions do not disrupt the overall antigenic structure of the hGHbp. This highresolution functional analysis will complement structural studies and provides a powerful basis for evaluating and engineering the energetics of hormone-receptor interactions. Moreover, the hormone-binding determinants identified here may be similarly located in other, homologous, receptors.Human growth hormone (hGH) is homologous to a large family of hormones that includes prolactins, placental lactogens, and proliferins (for review see ref. 1). Collectively, these hormones regulate a vast array of physiological effects, including growth, lactation, differentiation, and electrolyte balance (for reviews see refs. 2 and 3). These biological effects begin with the binding of hormone to specific cellular receptors. Systematic mutational studies have revealed functionally important residues in hGH for binding to the hGH receptor (4-6). In contrast, virtually nothing is known of the hormone-binding determinants in the hGH receptor.The hGH receptor cloned from liver (7) consists of a single polypeptide chain (620 residues total) containing an extracellular hormone-binding segment (246 residues), a single transmembrane region (23 residues), and a cytoplasmic segment (351 residues). The extracellular portion of the hGH receptor is found naturally in the blood stream (8) as an hGH-binding protein (hGHbp). Recent comparative sequence analyses suggest that the hGHbp is structurally related to a large family of cytokine receptors (9-11).The hGHbp (containing residues 1-238) has been expressed in Escherichia coli in large quantities (12), and it retains the same high affinity for hGH as its natural glycosylated counterpart. Here, using a combination of mutational and biophysical analysis, we have identified important hormone-binding determinants in the hGHbp. These determinants are chemically complementary to those previously identified in hGH, and they map predominantly to a cysteinerich region of the receptor and to loop regions on one side of a structural model proposed for cytokine receptors (10). This mutational strategy should be applicable to probing other hormone-receptor and protein-protein interactions for which little or no structural informa...

Mutagenesis Studies of the Human Erythropoietin Receptor

“…Several receptors in the cytokine receptor family have residues important for ligand binding located in the loop between the BЈ and CЈ ␤-strands (5,12,14). We have reported previously that Ser 152 in the BЈ-CЈ loop of the EPOR may have a role in EPO binding, based on the 16-fold increase in IC 50 value of the S152A-EBP (15).…”

“…Single alanine substitutions in the C-D loop of the GHbp had only marginal effects on GH binding (14). However, alanine substitutions of residues within the C-D loop of the human IL-5R␣ (D55, Y57) abolished IL-5 binding (27).…”

“…Support for these results was provided by a molecular model of the GM-CSF receptor complex (ligand, ␣-chain, and ␤ c ) in which the binding determinants identified by mutagenesis were predicted to be within bonding distance of each other (13). Alanine substitution mutagenesis has also been used to identify important ligand binding determinants in the extracellular domain of the growth hormone receptor (14), and many * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.…”

Systematic Structure-Function Analysis of the Small GTPase Arf1 in Yeast

“…However, large portions of Arf1p, including those determined by crystallography to bind other proteins, have not been tested directly for their function in cells. In this study we have used a systematic clustered charge-to-alanine mutagenesis approach (Bass et al, 1991;Bennett et al, 1991;Gibbs and Zoller, 1991) to generate mutations widely distributed on the surface of Arf1p in yeast. This approach is unbiased by expectations based on structure or sequence homology to other GTPases and has been used successfully in the related Rho-family GTPase CDC42 to discover novel phenotypes and to identify regions essential for cellular function (Kozminski et al, 2000).…”

The growth hormone receptor gene mutation of a japanese patient with laron syndrome

“…In many tissues, GHR generates two different mRNAs by an alternative splicing of exon 3, one for a full length receptor and the other for an isoform that lacks exon 3 (Urbanek et al, 1992;Sobrier et al, 1993;Mercado et aL, 1994). Although previous studies suggested that exon 3 is not necessary for GH binding, the functional significance of the splice variant without exon 3 remains unknown (Mercado et al, 1994;Bass et al, 1991). Several different GHR mutations have been reported among Laron syndrome patients of different ethnic origins.…”