K317, R319, and E320 within the proximal C-terminus of the bradykinin B2 receptor form a motif important for phospholipase C and phospholipase A2 but not connective tissue growth factor related signaling.
We showed previously that large domain exchanges between the bradykinin B2 (BKB2) and angiotensin II type 1a (AT1a) receptors can result in functional hybrids. However, when we proceeded to exchange the entire bradykinin B2 receptor (BKB2R) C-terminal tail with the AT1aR C-terminus, the hybrid, while continuing to bind BK and be endocytosed as wild type (WT) BKB2R, lost much of its ability to activate phosphatidylinositol (PI) turnover or the release of arachidonic acid (ARA). In this study, we constructed chimeric receptors within the proximal C-terminus between the BKB2R and AT1aR or bradykinin B1 receptor (BKB1R). The mutant and WT receptor cDNAs were stably transfected into Rat-1 cells. Also, point mutations were generated to evaluate the role of the individual residues within this region. These chimeric studies revealed that the proximal portion of the BKB2R C-tail is crucial for G protein-linked BKB2R functions. This region could not be swapped with the AT1aR to obtain a BK activated PI turnover or ARA release. Further studies demonstrated that the distal portion (325-330) of this region is exchangeable; however, the middle portion (317-324) is not. Small motif exchanges within this section identified the KSR and EVY motifs as crucial for G(alphaq), G(alphai) related signaling of the BKB2R. Point mutations then showed that the charged amino acids K317, R319, and E320 are the residues critical for linking to PI turnover and ARA release. However, these proximal chimeras showed normal receptor uptake. Interestingly, while apparently not activating G protein-linked signaling, the proximal tail AT1aR exchange mutant and the entire C-terminus exchange hybrid continued to cause a substantial bradykinin effected increase in connective tissue growth factor (CTGF) mRNA level, as WT BKB2R.
Yu
Jun
J
Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Liu
Bin
B
Eramian
David
D
Mierke
Dale
D
Taylor
Linda
L
Polgar
Peter
P
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D055513:Connective Tissue Growth Factor; D004705:Endocytosis; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010716:Phosphatidylinositols / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D044140:Receptor, Angiotensin, Type 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043783:Receptor, Bradykinin B1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043782:Receptor, Bradykinin B2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D010738:Type C Phospholipases / Q000378:metabolism*
C526316:CCN2 protein, rat; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D010716:Phosphatidylinositols; D012333:RNA, Messenger; D044140:Receptor, Angiotensin, Type 1; D043783:Receptor, Bradykinin B1; D043782:Receptor, Bradykinin B2; D011993:Recombinant Fusion Proteins; D055513:Connective Tissue Growth Factor; D016718:Arachidonic Acid; D018698:Glutamic Acid; D001120:Arginine; D010741:Phospholipases A; D054467:Phospholipases A2; D010738:Type C Phospholipases; D008239:Lysine