Explanatory power of the Hill slope in radioligand saturation and competition binding

4.1.1.1 Indicators of cooperativity and ligand–receptor stoichiometry

Estimation of the inflection points of semilogarithmic binding curves provides information on ligand affinities (Fig. 4.1.2 A). It is assumed that binding has reached equilibrium, thereby obeying the law of mass action and that ligand-receptor interactions occur ac-cording to a 1:1 stoichiometry. Therefore, the Hill slope (n_H) is usually adjusted to unity and the competition curve between 10% and 90% displacement covers approx. a hun-dredfold range of ligand concentrations (between 0.1- and 10-fold·IC₅₀; cf. Lazareno, 2001), as depicted in Fig. 4.1.2 B (“normal" curve is linear between approx. 20 and 80%

displacement). However, algorithms for fitting experimental data by a four-parameter logistic curve with a variable slope often yield a slope different from unity (Prinz and Schönichen, 2008). In such cases, curves do not cover 10–90% occupancy over a range of 2 log units (Lazareno, 2001; cf. shallow and steep curves in Fig. 4.1.2 B).

Fig. 4.1.2. Pharmacologic parameters derived from the analysis of competition binding curves:

(A) the inflection point (IC50) and (B) Hill slope nH (adopted from Limbird, 2004 with modifications).

A B

Table 4.1.1. Various possible explanations for nH ≠ 1 (cf. Repke and Liebmann, 1987).

The value of nH Possible explanations 0.5 Heterogenic ligand binding sites

Negative cooperativity

1 Homogenic population of ligand binding sites

2 Positive cooperativity

Derived from the Hill equation, the Hill slope corresponds to the number of ligand mole-cules interacting with a receptor in an “all or none” reaction, and therefore it might reflect the actual ligand-receptor stoichiometry (cf. Eq. 4.1.1–2).

R A R

nA   _n (Eq. 4.1.1)

   

nH

max A K

A B

B

 

(Eq. 4.1.2) Depending on the position of the binding pockets for the two pharmacophoric moieties of a twin compound on a single receptor molecule, two independent receptors or a receptor dimer, the value of n_H could vary between 0.5 and 2, as shown in Fig. 4.1.3. Neverthe-less, apart from the ligand-receptor stoichiometry, other important factors should be con-sidered as well, for example cooperativity (Table 4.1.1; cf. Repke and Liebmann, 1987).

Fig. 4.1.3. Possible spatial arrangements of ligands (mono- or bivalent) and receptors (a single receptor, two non-linked receptors or a conformationally linked receptor dimer). The values of Hill slopes correspond to the number of ligand molecules interacting with a receptor in an “all or none”

reaction (Eq. 4.1.1). The classical situation with the 1:1 ligand-receptor stoichiometry is high-lighted by a red box.

Introduction 77 4.1.1.2 Biphasic Scatchard plots

The analysis of saturation binding curves might also elucidate the binding mode of a ra-dioligand. According to Scatchard, in case of a competitive ligand-receptor interaction with 1:1 stoichiometry, the classical linearizing transformation of the radioligand satura-tion curve gives a single straight line, as shown in Fig. 1.3.1 B, where the dissociasatura-tion constant K_d equals the negative inverse of the slope (Lazareno, 2001). However, a pre-vious study by Monczor et colleagues on U-937 cells, using [³H]TIO as the radioligand, revealed a convex (biphasic) shape of the Scatchard plot (Fig. 4.1.4 A). This phenome-non might be explained either by incorrectly determined unspecific binding (competitor concentration too low) or by the existence of binding sites with different affinity for the radioligand (Repke and Liebmann, 1987). Indeed, low and high affinity binding sites were found for [³H]TIO, with the high-affinity binding site disappearing in the presence of 10 µM GTPγS. Thus, binding of TIO to H₂Rs present in the free and in the G-protein-coupled form is a plausible explanation of these results. The obvious preference of TIO for G-protein-bound receptors indicates an inverse agonism of TIO, as predicted by the extended ternary complex model (cf. Fig. 1.1.6), assuming the existence of two classes of interconverting binding sites (Fig. 4.1.4 B; cf. Monczor et al., 2003).

Fig. 4.1.4. Biphasic Scatchard plots: (A) saturation binding of [³H]TIO at the H2R in U-937 cells.

Purified fractions of U-937 membranes were incubated for 2 hours (adopted from Monczor et al., 2003 with modifications). (B) Two-site model of ligand-receptor interactions. The slopes of the red lines equal the negative inverses of the affinity constants of high-affinity (KdH) and low-affinity (KdL) binding sites (adopted from http://www.chemistry.emory.edu/justice/test/receptor_measurement.

htm with modifications).

N.B. Hill slopes of competition curves smaller than unity can also result from the exis-tence of two non-interconverting (ortho- and allosteric) binding sites on the same recep-tor molecule. The hypothesis of allosterism will be addressed in chapter 5.

A B

4.2 Materials and methods

4.2.1 Materials

Sf9 cells and high-titer baculovirus stocks, encoding H₂Rs, G_sαS , G_iα2, G_β1γ2 proteins as well as H₂R-G_sαS and hH₄R-GAIP fusion proteins, were kindly provided by Prof. Dr. Ro-land Seifert (Institute of Pharmacology, Medical School of Hannover, Germany). The generation of the baculoviruses encoding the hH₂R-G_sαS and the gpH₂R-G_sαS has been described elsewhere (Kelley et al., 2001).

The mono- and bivalent H₂R ligands (Fig. 1.2.4) were synthesized and provided by Dr.

Anja Kraus (Kraus, 2007; Kraus et al., 2009) and Mr. Tobias Birnkammer (cf. prospective doctoral thesis of T. Birnkammer). HIS was from Acros Organics (Geel, Belgium). RAN and FAM were obtained from Sigma (St. Louis, MO, USA). TIO was donated by Prof. Dr.

Sigurd Elz (Institute of Pharmacy, University of Regensburg, Germany). GTPγS (Fig.1.1.4) was from Roche Diagnostics (Mannheim, Germany). Radioactive tracers [³⁵S]GTPγS (1100 Ci/mmol), [³H]HIS (14.2 Ci/mmol) and [³H]TIO (77.2 and 82.2 Ci/mmol) were from PerkinElmer Health Sciences (Boston, MA, USA) and [³H]TIO (82.2 Ci/mmol) was from American Radiolabeled Chemicals (St. Louis, MO, USA). [³ H]UR-DE257 (69.3 Ci/mmol) and the red fluorescent H₂R ligands were synthesized by Dr.

Daniela Erdmann in our laboratory as described previously (Fig. 1.2.5; Erdmann, 2010).

Insect-Xpress medium for Sf9 cells was from Lonza (Verviers, Belgium). BSA was from Behringwerke (Marburg, Germany). FCS was obtained from Biochrom (Berlin, Ger-many). Gentamicin was from Lonza (Walkersville, MD, USA). Tris was purchased from USB (Cleveland, OH, USA). MgCl2 and EDTA were from Merck (Darmstadt, Germany).

Polyethylenimine (PEI) and phenylmethylsulfonyl fluoride (PMSF) were from Sigma-Aldrich (Steinheim, Germany). Benzamidine was from Acros Organics (Geel, Belgium).

Leupeptin was obtained from Gerbu Biotechnik (Gaiberg, Germany). DC protein assay kit was purchased from Bio-Rad Laboratories (Munich, Germany) Round-bottom poly-propylene test tubes (4 mL) were from Kabe Labortechnik (Nümbrecht, Germany). Nee-dles Neolus were from VWR International (Ismaning, Germany). GF/B glass-fibre filters were purchased from Brandel (Gaithersburg, MD, USA). The 6-mL scintillation counting vials were obtained from Sarstedt (Nümbrecht, Germany). Scintillation cocktail Rotiszint eco plus was from Carl Roth (Karlsruhe, Germany). High purity water, used in all assays, was prepared with Milli-Q Reagent Water System from Millipore (Molsheim, France).

Materials and methods 79 The human promonocytic U-937 cell line was a kindly provided by Prof. Dr. Roland Seifert. CHO-rH₂R-A₂ cells (Traiffort et al., 1992) were a gift by Dr. Jean-Michel Arrang (Institut National de la Santé et de la Recherche Médicale, Paris, France). The genera-tion of CHO-hH₂R-G_α16 cells was described before (Schneider, 2005). CHO cells were from the DSMZ (Deutsche Sammlung für Mikroorganismen und Zellkulturen, Braunschweig, Germany). The pcDNA3.1(+)-Neo-hH4R plasmid was from the Guthrie cDNA Resource Center (Sayre, USA; cf. Mosandl, 2009). The jetPEI transfection re-agent, from Polyplus-transfection (Illkirch, France), was donated by Peqlab Biotechnolo-gie (Erlangen, Germany). DMEM and Ham’s F12 nutrient mixtures were purchased from Sigma-Aldrich. FCS and geneticin (G418) were from Biochrom (Berlin, Germany). Hy-gromycin B was obtained from Mobitec (Göttingen, Germany). Trypsin-EDTA (10x) was from PAA Laboratories (Pasching, Austria). Leibovitz' L-15 medium, without phenol red, was from Invitrogen (Karlsruhe, Germany). Transparent 6-well and 96-well plates were purchased from Greiner (Frickenhausen, Germany), whereas transparent 24-well plates were from Becton Dickinson (Heidelberg, Germany). Safety-Multifly needles (21G) and 4-mL S-Monovettes (for blood collection by venous puncture) were from Sarstedt. Lym-phoprep solution was from Axis-Shield PoC (Oslo, Norway).

All experimental data were analyzed with the Prism 5 program (GraphPad Software, San Diego, CA, USA).