Effects of intracellular tyrosine residue mutation and carboxyl terminus truncation on signal transduction and internalization of the rat bradykinin B2 receptor.
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 phenylalanine-containing 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 [3H]bradykinin with essentially the same Kd of approximately 2.2 nM as cells transfected with the wild type bradykinin B2 receptor. However, mutating Tyr-131 resulted in important changes in bradykinin-stimulated phosphoinositide turnover and arachidonate release. For example, exchanging Tyr-131 for alanine led to an 80% decreased arachidonate release (p < 0.005), 90% decrease in inositol phosphate (IP) accumulation (p < 0.001), with receptor uptake at 15 min remaining essentially unchanged. Mutating the same Tyr to phenylalanine resulted in unchanged bradykinin-stimulated IP accumulation, only a slightly lowered arachidonate release, and unchanged receptor uptake at 15 min. Mutating Tyr-321 to alanine resulted in a very different pattern. There was a small but significant reduction in arachidonate release (p < 0.03) and IP accumulation (p < 0.008) with a large, 30%, increase in receptor uptake at 15 min (p < 0.010). Truncation of a portion of the carboxyl tail also proved meaningful, with a 60% decrease in arachidonate release and an 80% decrease in IP accumulation. The truncation also resulted in a large, 130%, decrease in receptor uptake at 15 min (p < 0.023). Taken together, these results point to Tyr-131 as an important element in determining bradykinin-stimulated arachidonate release and IP accumulation. Tyrosine phosphorylation at this site apparently does not play a major role. Tyr-131, Tyr-321, and the carboxyl tail appear to be important in determining receptor uptake.
Prado
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GN
Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Taylor
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L
Polgar
P
P
D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001920:Bradykinin / Q000494:pharmacology*; D017124:Conserved Sequence; D004705:Endocytosis; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017433:Protein Structure, Secondary; D051381:Rats; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000096:biosynthesis / Q000737:chemistry* / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D015398:Signal Transduction; D014443:Tyrosine
D007295:Inositol Phosphates; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D011994:Recombinant Proteins; D016718:Arachidonic Acid; D014443:Tyrosine; D001920:Bradykinin