- Original Articles
- Open Access
Construction of a Novel Bifunctional Biogenic Amine Receptor by Two Point Mutations of the H2-Histamine Receptor
© Molecular Medicine 1995
- Published: 1 March 1995
H2-histamine receptors mediate a wide range of physiological functions extending from stimulation of gastric acid secretion to induction of human promyelocyte differentiation. We have previously cloned the H2-histamine receptor gene and noted that only three amino acids on the receptor were sufficient to define its specificity and selectivity. Despite only modest overall amino acid homology (34% amino acid identity and 57.5% similarity) between the H2-histamine receptor and the receptor for another monoamine, the β2-adrenergic receptor, there is remarkable similarity at their critical ligand binding sites. We hypothesized that, if the specificity and selectivity of both receptors are invested in just three amino acids, it should be possible to convert one of the receptors into one that recognizes the ligand of the other by simple mutations at only one or two sites.
Material and Methods
We explored the effect of two single mutations in the fifth transmembrane domain of the H2-histamine receptor, which encompasses the sites that determine H2 selectivity. The canine H2 receptor gene was mutated at Asp186 and Gly187 (Asp186 to Ala186 and Gly187 to Ser187) by oligonuceotide directed mutagenesis. The coding region of both the wild-type and mutated H2 receptors was subcloned into the eukaryotic expression vector, CMVneo, and stably transfected into Hepa cells and L cells. The biological activity of histamine and epinephrine on the expressed receptor was examined by measurement of cellular cAMP production and inositol trisphosphate formation.
Hepa cells transfected with the Ala186-Ser187 mutant H2 receptor demonstrated a biphasic rise in cAMP in response to epinephrine with an early phase (ED50 ≈10−11 M) that could be inhibited by both propranolol and cimetidine. Epinephrine also induced IP3 generation in the same cells, a biological response that is characteristic of activation of the wild-type H2 but not of the β-adrenergic receptor. L cells transfected with the Ala186-Ser187 mutant H2 receptor also responded to epinephrine in a cimetidine and propranolol inhibitable manner.
We converted the H2-histamine receptor into a bifunctional one that has characteristics of both histamine and adrenergic receptors by two simple mutations. These results support the hypothesis that ligand specificity is determined by only a few key points on a receptor regardless of the structure of the remainder of the molecule. Our studies have important implications on the design of pharmacological agents targeted for action at physiological receptors.
The availability of genes encoding an increasingly wide array of receptors for regulatory substances has led to insight into the molecular basis of ligand-receptor interactions. Recently, we cloned the H2-histamine receptor gene (1) and noted that only three amino acids on the receptor were sufficient to define the specificity and selectivity of the receptor (2). By extrapolation of these findings, it is conceivable that ligand specificity is determined by only a few key points on a receptor regardless of the structure of the remainder of the molecule. We have previously noted a remarkable similarity between the H2-histamine receptor and the receptor for another monamine, the β2-adrenergic receptor, at their critical ligand binding sites. However, there is otherwise only modest overall amino acid homology (34% amino identity and 57.5% similarity), and, furthermore, there is no evidence to indicate cross-reactivity of histamine at β2-adrenergic receptors or, conversely, catecholamines at H2-histamine receptors. We reasoned that, if our hypothesis that the specificity and selectivity of both receptors are invested in just three amino acids is correct, it should be possible to convert one of the receptors into one that recognizes the ligand of the other by simple mutations at only one or two sites. In the present studies, we provide support for this hypothesis by demonstrating that two amino acid substitutions are sufficient to convert the H2-histamine receptor into a novel bifunctional receptor that has characteristics of both histamine and adrenergic receptors.
Mutagenesis and Expression
For our studies, the canine H2-histamine receptor gene was subcloned into the sequencing vector M13 and used as a template for the synthesis of DNA with specific mutations according to the method of Kunkel (5). A polymerase chain reaction (PCR) strategy was used to subclone the coding region of both the wild-type and mutated H2-histamine receptors into the eukaryotic expression vector CMVneo, as previously described (2,6). Hepa cells (derived from a rat hepatoma) and L cells (derived from a mouse fibroblast-like cell) were then transfected with the receptor/CMVneo constructs by calcium phosphate copre-cipitation (7) and clones were selected in media containing the neomycin analogue G-418. The expression of receptor RNA was examined by Northern blot analysis and clones expressing roughly equivalent amounts of receptor RNA were used for further studies.
Intracellular cyclic 3′,5′-adenosine monophosphate (cAMP) accumulation was measured using a cAMP Assay Kit (TRK 432; Amersham, Arlington Heights, IL, U.S.A.). Cells transfected with the receptor genes were grown to confluence in 12-well (2.4 × 1.7 cm) tissue culture plates. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY) containing 4.5 g/100 ml glucose, 10% fetal calf serum, 100 units/ml penicillin and streptomycin, 1 mM sodium pyruvate and 1 mg/ml of geneticin. For assays, this media was removed and cells were washed twice with Earle’s balanced salt solution (EBSS) containing 10 mM HEPES (pH 7.4), 1 mM glutamine, 26.5 mM sodium bicarbonate, and 100 mg/ml bovine serum albumin. An aliquot (0.5 ml) of EBSS was placed into each well along with 5 µl of 2 × 10−2 M isobutylmethylxanthine. Varying concentrations of agonist were added and the cells were incubated for 30 min at 37°C. Ice-cold 30% trichloracetic acid (500 µl/well) was added to stop the reaction and precipitate cellular protein. The cells were scraped and transferred to 16 × 150 mm glass tubes then placed on ice for 30 min. The precipitate was then centrifuged for 10 min at 1,900 × g and the supernatant was ether extracted, lyophilized, and resuspended in 50 mM tris, 2 mM EDTA (pH 7.5). cAMP content was then measured by competitive binding assay according to the assay instructions.
Inositol Phospholipid Assays
Transfected Hepa cells were grown to confluence in 2.4 × 1.7 cm multiwell plates and prelabeled with myo [2-3H] inositol at 37°C for 2 hr, adding LiCl (10 µM) 10 min before completing the preincubation period. Histamine or epinephrine were added for varying time intervals and the water soluble cellular products were extracted and separated by ion exchange chromatography on Dowex-1 resin columns as previously described using 100 mM increments of ammonium formate (8). Inositol monophosphate was eluted with 10 mM formic acid/100 mM ammonium formate/5 mM disodium tetraborate, inositol bisphosphate was eluted with 20 mM formic acid/200 mM ammonium formate/5 mM disodium tetraborate, and inositol trisphosphate (IP3) was eluted with 30 mM formic acid/300 mM ammonium formate/5 mM disodium tetraborate. The fraction containing IP3 consists of a mixture containing the 1,4,5 and the 1,3,4 isomers.
We have demonstrated that two amino acid substitutions are sufficient to convert the H2-histamine receptor into a bifunctional one that has characteristics of both the histamine and adrenergic receptors. Our studies suggest that the ligand specific binding properties of a receptor are determined by a surprisingly limited number of key amino acids in its molecular structure. Although there is broad structural similarity among seven transmembrane G protein-linked receptors, there is relatively low amino acid homology between families of receptors for different classes of agonists. Despite the relatively modest overall structural homology between the H2-histamine and β2-adrenergic receptors, two simple amino acid substitutions are sufficient to convert the former receptor into one that has the ligand recognition characteristics of both. Seemingly minor amino acid changes have been reported previously to have profound effects on the pharmacology of G protein-linked seven transmembrane receptors. For example, the alteration of a single amino acid has been shown to account for the pharmacological differences between the rat and human 5-hydroxytryptamine 1B receptors (10,11) and the canine and human cholecystokinin-B/gastrin receptors (12). Similarly, a point mutation in the α2-adrenergic or 5-hydroxytryptamine 1A receptors changes their affinity for β-adrenergic receptor antagonists (13,14). In other studies the receptors for the tachykinins substance P (NK1 receptor) and neurokinin B (NK3 receptor) have been shown to exhibit alterations in receptor binding affinity to nonpeptide antagonists as a result of changes in only one or two of its amino acid residues (15–17).
Our experiments provide the first demonstration that such minor amino acid changes can result in changes in ligand specificity so major that they can cross the gap between entire families of receptors that have only modest structural homology. These findings have important implications in the design of pharmacological agents that are directed at physiological receptors. It is possible, for example, that so-called specific receptor antagonists or agonists may have crossover effects on other groups of receptors that would not be predicted on the basis of overall structural homology. Moreover, it may be possible to utilize unique agonists or antagonists for targeting ligand delivery to artificially constructed mutant receptors that are transfected into various tissues in the body.
This work was supported by National Institutes of Health (NIH) Grants RO1DK34306 and RO1DK47434, and funds from the University of Michigan Gastrointestinal Peptide Research Center (NIH Grant P30DK34933). Dr. Gantz is a recipient of a Veterans Administration Research Associate Award. We thank Drs. J. Dixon and M. Uhler for their helpful comments on this manuscript.
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