Possible role for ligand binding of histidine 81 in the second transmembrane domain of the rat prostaglandin F_2α receptor

Abstract

For the five principal prostanoids PGD P , PGE P , PGF PK , prostacyclin and thromboxane A P eight receptors have been identified that belong to the family of G-protein-coupled receptors. They display an overall homology of merely 30%. However, single amino acids in the transmembrane domains such as an Arg in the seventh transmembrane domain are highly conserved. This Arg has been identified as part of the ligand binding pocket. It interacts with the carboxyl group of the prostanoid. The aim of the current study was to analyze the potential role in ligand binding of His-81 in the second transmembrane domain of the rat PGF PK receptor, which is conserved among all PGF PK receptors from different species. Molecular modeling suggested that this residue is located in close proximity to the ligand binding pocket Arg 291 in the 7th transmembrane domain. The His81 (H) was exchanged by sitedirected mutagenesis to Gln (Q), Asp (D), Arg (R), Ala (A) and Gly (G). The receptor molecules were N-terminally extended by a Flag epitope for immunological detection. All mutant proteins were expressed at levels between 50% and 80% of the wild type construct. The H81Q and H81D receptor bound PGF PK with 2-fold and 25-fold lower affinity, respectively, than the wild type receptor. Membranes of cells expressing the H81R, H81A or H81G mutants did not bind significant amounts of PGF PK . Wild type receptor and H81Q showed a shallow pH optimum for PGF2K K binding around pH 5.5 with almost no reduction of binding at higher pH. In contrast the H81D mutant bound PGF PK with a sharp optimum at pH 4.5, a pH at which the Asp side chain is partially undissociated and may serve as a hydrogen bond donor as do His and Gln at higher pH values. The data indicate that the His-81 in the second transmembrane domain of the PGF PK receptor in concert with Arg-291 in the seventh transmembrane domain may be involved in ligand binding, most likely not by ionic interaction with the prostaglandin's carboxyl group but rather as a hydrogen bond donor.z 1999 Federation of European Biochemical Societies.

Structural Differences of Bacterial and Mammalian K+Channels

“…The difference in affinity between wt KTX at high pH o and H34A might be caused by an interaction between His 34 /KTX and residues of the channel via the nitrogen atoms, which is destroyed by the substitution with alanine. More conservative exchanges of His 34 with glutamine or asparagine might have the same blocking strength as unprotonated wt KTX, as shown for several histidine interactions in other proteins (28,29).…”

Ionization Properties of Histidine Residues in the Lipid Bilayer Membrane Environment

“…A further example is provided by residues 1-19 of the islet amyloid polypeptide (IAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] ), for which the rat and human sequences are identical, except that residue Arg 18 in rat is replaced by His 18 in human (40). At pH 7.3, the more toxic human IAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] with His 18 (presumably neutral) inserts more deeply into lipid micelles, whereas the less toxic rat IAPP [1][2][3][4][5][6][7][8][9][10][11]…”

“…It is therefore vitally important to also know the ionization properties of the His side chain at various locations within lipid bilayer membranes. Indeed, as with soluble proteins, many membrane proteins contain functionally important His residues within their transmembrane domains: for example, those of the photosynthetic reaction center (4), cytochrome c oxidase (5,6), influenza A M2 channel (7)(8)(9)(10), and other transporters and channels (11)(12)(13)(14). Given that proton conduction may require clusters of multiple residues (5,6), it is important to know the limits for the titration properties of candidate residues such as histidines and carboxyl groups, among others, in bilayer membranes.…”

Identification of Determinants of Ligand Binding Affinity and Selectivity in the Prostaglandin D2 Receptor CRTH2

“…Sequence comparison suggests that the CRTH2 receptor differs in the manner in which it binds ligand compared with the other prostanoid receptors. For instance, studies utilizing DP/IP receptor chimeras and mutants have suggested that high affinity binding of PGD 2 by the mouse DP receptor requires Lys-75 in TM II (32), and His-81 at the equivalent position in the rat FP receptor has been suggested to play a role in binding PGF 2␣ (35). For the CRTH2 receptor, the corresponding residue, alanine (mouse and rat) or serine (human), is unlikely to participate in ligand binding in a similar manner.…”