Effects of Intracellular Tyrosine Residue Mutation and Carboxyl Terminus Truncation on Signal Transduction and Internalization of the Rat Bradykinin B2 Receptor

Abstract

Presently, little is known of the amino acid motif(s) participating in bradykinin B2 receptor-mediated signal transduction processes. In this report we investigate the potential role of the two existing tyrosine (Tyr) residues in the intracellular regions and the carboxyl terminus in the regulatory function of this receptor. Rat-1 cells, which do not contain detectable bradykinin B2 receptor, were transfected with wild type and mutant receptor cDNAs. Tyr-131 and Tyr-321 were each mutated to corresponding alanine-, serine-, and phenylalaninecontaining sequences. The last 34 amino acid residues of the carboxyl terminus were truncated. Rat-1 cells transfected with the mutant forms of the receptor cDNA including the truncated COOH-terminal cDNA all bound Bradykinin is a nine-amino acid peptide hormone exerting diverse biological action ranging from a role in the inflammatory process to regulatory effects on vascular permeability, blood pressure, generation of pain, and renal homeostasis (1, 2). Bradykinin expresses its physiological effect through activation of a B2 type receptor. The bradykinin B2 receptor cDNA has been isolated from several species such as mouse, rat, and human (3-5). The receptor is G-protein-coupled with a structure characterized by seven transmembrane regions (4). Upon binding, the receptor activates a number of enzyme systems involved in signal transduction including phospholipase C and cytosolic phospholipase A 2 (6). When stably transfected into such cells as hamster CCL39 the receptor retains its signal paths (7).Investigation of the regulatory regions or domains of the bradykinin B2 receptor which participate in the activation of second messenger responses has been hampered by the unavailability of immunoprecipitating antibodies against the receptor. Herein we target single amino acid and amino acid sequence moieties that could prove crucial to the function of this receptor in signal transduction. These include the two existing tyrosines at positions 131 and 321 and the carboxyl terminus consisting of 34 amino acid residues including four serines and two threonines. We analyze the ability of normal and mutant bradykinin B2 receptors to stimulate inositol phosphate (IP) 1 production and release of arachidonate. Our results show that tyrosine (Tyr) 131 and 321 and the carboxyl terminus participate variably in function of both paths as well as in receptor internalization. Cell Culture-Rat-1 cells were obtained from Dr. Robert Weinberg (Whitehead Institute, MIT) as a generous gift. Cells were grown at 37°C in a humidified atmosphere with 5% CO 2 in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum supplemented with 50 units/ml penicillin and 50 g/ml streptomycin. All tissue culture dishes were obtained from Fisher Scientific. EXPERIMENTAL PROCEDURES Materials-[Site-directed Mutagenesis-The overlap extension polymerase chain reaction method was used to generate mutants of the bradykinin B2 receptor utilizing as template the rat bradykinin B2 cDNA clone in the vector pRC/CMV (I...

Bradykinin-induced Internalization of the Human B2Receptor Requires Phosphorylation of Three Serine and Two Threonine Residues at Its Carboxyl Tail

“…These observations are in agreement with those of Faussner et al (7) who reported that truncations either at Gly 327 or Lys 315 diminished the capacity to internalize BK but left the capacity to activate PLC unchanged. These and our results are, however, inconsistent with a recent report (39) claiming compromised ligand internalization associated with reduced PLC activity for a rat B 2 receptor mutant devoid of the terminal 34 residues (corresponding to tR331 for the human receptor). Differences in species (rat versus man) and expression systems (Rat-1 versus COS-7, CHO-K1, and HEK 293) may help explain some of the observed discrepancies.…”

“…This contrasts with the findings that the human ␤ 2 -adrenergic receptor truncated at position 365 was internalized to a greater extent than the wild type receptor in these cells (14). Our data show that the COOH-terminal tail contributes significantly to the internalization of the B 2 wt, although other structures such as the intracellular loops may also be involved, albeit with a reduced efficiency (39).…”

The Proximal Portion of the COOH Terminus of the Oxytocin Receptor Is Required for Coupling to Gq, but Not Gi

“…Depending on the particular GPCR, these activities include G protein coupling (3-7), selectivity of G protein isotype (11, 11-14, 45), and homologous desensitization or internalization (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). Whereas truncation of some GPCRs has been shown to modify any one or more of its functions, shortening of the COOH terminus of other family members has no apparent effects (8 -10).…”

“…Work on the ␤-adrenergic receptor, in particular, has indicated that one mechanism of desensitization involves the rapid internalization of membrane-bound receptors following agonist stimulation (15). Removal of the COOH-terminal tail of some GPCRs has been shown to greatly reduce agonist-induced internalization of the receptor while having little or no effect on signal transduction (21)(22)(23)(24)(25).…”

Ace inhibitors - activators of kinin receptors

“…В последнее десятилетие в рецепторах кининов были идентифицированы аминокислотные остатки, важные для взаимодействия с G-белком и дальнейшей передачи сигнала. Было показано, что усечение С-концевой последовательности в Б 2 Р человека вплоть до Ser 316 ослабляет, но полностью не тормозит сопряжение рецептора с Ga q -белком [74][75][76]. С другой стороны, усечение выше этого аминокислотного остатка полностью уничтожает активность Б 2 Р, что возможно приводит к разрыву a-cпирали (a8), включающей аминокислотные остатки 310 -321, которая, по-видимому, требуется для эффективного сопряжения рецептора с Ga q -белком (рис.…”