The luteinizing hormone receptor: influence of buffer composition on ligand binding and signaling of wild type and mutant receptors

Abstract

There is evidence that ligand binding to and ligand-mediated signaling by the luteinizing hormone receptor (LHR) are influenced by buffer conditions, including ionic type and strength, an issue that becomes important in comparing functional parameters obtained on receptor mutants under different conditions. In order to study this phenomenon, we performed binding (kinetic and saturation) and signaling studies of human chorionic gonadotropin (hCG) with wild type (wt) LHR and several mutants expressed in COS-7 cells using two common buffer systems. One buffer was of low ionic strength and contained a low concentration of Na⁺, while the other had a near-physiological concentration of Na⁺. Emphasis was placed on mutations of two amino acid residues in the hinge region of the ectodomain (E332 and D333). It was found that the buffer of higher ionic strength, primarily from Na⁺, led to an increase of about 4-fold in the K_d of hCG binding to wt and mutant LHRs. The reduced binding affinities were attributable to a comparable reduction in the rate constants of association, with no significant differences in the calculated rate constants of dissociation in the two buffers. Analysis of the signaling properties of these mutants showed that, when corrected for the amount of hCG bound under the conditions of the signaling assay, the maximal ligand-mediated cAMP produced in cells maintained in the buffer of low ionic strength was comparable for wt LHR and the mutants, only the D333A mutant being somewhat elevated. In the buffer of higher ionic strength, however, the response by wt LHR was significantly greater than that of the mutants. These results show that E332 and D333 are important in hormone-mediated signaling, but only in the buffer of higher Na⁺ concentration. In addition to mutants of these two residues, the buffer of higher ionic strength also led to reduced binding to a number of mutants throughout the receptor. Since these mutants included additional replacements in the ectodomain and transmembrane helices 6 and 7, the general nature of the buffer effect on wt and mutant LHRs suggests that electrostatic effects are contributing to ligand binding and/or that the LHR ectodomain may exist in two conformations, one being more accessible to ligand at reduced ionic strength.

Introduction

The luteinizing hormone receptor (LHR) is a member of the G protein-coupled receptor family characterized by a relatively large N-terminal ectodomain responsible for high-affinity ligand binding (Ascoli et al., 2002). LHR and the other two members of the glycoprotein hormone receptor subfamily, follicle-stimulating hormone receptor (FSHR) and thyroid-stimulating hormone receptor (TSHR), contain an invariant ten amino acid residue sequence in their ectodomains in close proximity to the first transmembrane helix: FNPCEDIMGY. Earlier mutagenesis studies from our laboratory suggested that Glu-332 and Asp-333, located within this conserved region, were important in rat LHR signaling, expression, or stability, but not ligand binding (Huang and Puett, 1995, Alvarez et al., 1999). Moreover, a naturally occurring homozygous mutation in human LHR, leading to a replacement of the invariant Glu with Lys, was shown to lead to primary amenorrhea and male pseudohermaphroditism in affected 46XX and 46XY individuals, respectively (Stavrou et al., 1998).

A more recent comparative study with wild type (wt) and mutant forms of LHR, FSHR, and TSHR, containing replacements of Glu→Ala, Asp, of Asp→Ala, Glu, and of Glu-Asp→Asp-Glu, showed differential responses in binding and signaling in two commonly utilized buffer systems for in vitro assays (Angelova and Puett, 2002). For example, ligand-mediated signaling of LHR mutants at these two residues was dependent on the two buffer systems used. One major difference in the two buffer systems is that of ionic strength, influenced largely by the concentration of Na⁺. While there is one report that Na⁺ reduces the rate constant of association of human chorionic gonadotropin (hCG) binding to LHR (Combarnous et al., 1986), others have not found an effect of Na⁺ on hCG–LHR equilibrium binding (Buettner and Ascoli, 1984, Quintana et al., 1993). There is, however, a significant reduction in the affinity of ovine LH binding to LHR (Buettner and Ascoli, 1984). In contrast to the absence of an effect of Na⁺ on hCG binding to LHR, it has been reported that the addition of Na⁺ to cell media reduces the basal production of cAMP (Quintana et al., 1993, Cetani et al., 1996), suggesting that Na⁺ may constrain LHR in a more inactive form. Cells expressing TSHR also exhibit reduced basal production of cAMP in Na⁺-containing buffers (Cetani et al., 1996). In addition to LHR, there are reports showing pronounced effects of cations and ionic strength on binding and signaling of the cognate glycoprotein hormone to FSHR (Anderson and Reichert, 1982) and TSHR (Tramontano and Ingbar, 1986). Numerous other G protein-coupled receptors are well documented to be influenced by the concentration of extracellular Na⁺ (Horstman et al., 1990, Martin et al., 1999, Neve et al., 2001).

In view of these various reports on the influence of Na⁺ and ionic strength on receptor function, we investigated the effect of two buffers frequently used in in vitro experiments on equilibrium and kinetic binding properties of hCG to wt rat LHR, the single mutants E332A, D and D333A, E, and the double (reversal) mutant E332D/D333E in transfected COS-7 cells, as well as ligand-mediated signaling. It was found that measurements in the buffer of increased ionic strength led to increased K_d and decreased rate constants of association, with no significant effect on the calculated rate constant of dissociation. Maximal ligand-mediated signaling of wt LHR was about 2-fold higher in the buffer of higher ionic strength and considerably lower for the mutant LHRs. When corrected for the degree of receptor occupancy, cAMP production remained greatest for wt LHR in the buffer of higher ionic strength (>20 pmol ml⁻¹ fmol⁻¹ hCG bound), was about 5–6 pmol ml⁻¹ fmol⁻¹ hCG bound in the buffer of lower ionic strength, and ranged between 4 and 12 pmol ml⁻¹ fmol⁻¹ hCG bound the single mutants in both buffers, and was significantly lower in the reversal mutant in both buffers.

These results suggest that higher ionic strength buffers provide electrostatic shielding, thus diminishing oppositely charged ligand-receptor interactions, and may favor a more closed form of the LHR ectodomain, both effects of which would reduce the rate of ligand association. Moreover, an important functional role of the Glu-Asp sequence is indicated: either of the acidic residues is sufficient for partial or maximal cAMP production by hCG at higher and lower ionic strengths, respectively, but a simple reversal greatly diminishes signaling, implying considerable specificity in E332 and D333 side chain interactions with other regions of the receptor.