Splice isoforms of alpha(1a)-adrenoceptor in rabbit.
Two splice isoforms of rabbit alpha(1a)-adrenergic receptor (AR), (named alpha(1a)-OCU.2-AR and alpha(1a)-OCU.3-AR) have been isolated from the liver cDNA library in addition to the previously reported isoform (alpha(1a)-OCU.1-AR). Although they have the identical splice position with human alpha(1a)-AR isoforms, the C-terminal sequences are distinct from those of human isoforms. Among these rabbit alpha(1a)-AR isoforms, there are no significant differences in pharmacological properties: high affinity for prazosin, WB4101, KMD-3213 and YM617 and low affinity for BMY7378, using COS-7 cells expressing each isoform by radioligand binding assay. Competitive reverse transcription-polymerase chain reaction (RT - PCR) analysis revealed that mRNA of alpha(1a)-ARs was expressed in liver, thoracic aorta, brain stem and thalamus of rabbit. The splice isoforms exhibited a distinct distribution pattern in rabbit; alpha(1a)-OCU. 1-AR was expressed most abundantly in those tissues. CHO clones, stably expressing each isoforms with receptor density 740 fmol mg(-1) protein in alpha(1a)-OCU.1-AR, 1200 fmol mg(-1) in alpha(1a)-OCU.2-AR and 570 fmol mg(-1) in alpha(1a)-OCU.3-AR, respectively, showed a noradrenaline-induced increase in inositol trisphosphate which was suppressed by prazosin. Noradrenaline elicited a concentration-dependent increase in extracellular acidification rate (EAR) in the CHO clones with pEC(50) values of 6. 19 for alpha(1a)-OCU.1-AR, 6.49 for alpha(1a)-OCU.2-AR and 6.58 for alpha(1a)-OCU.3-AR, respectively. Noradrenaline caused a concentration-dependent increase in intracellular Ca(2+) concentration ([Ca(2+)]i) in the CHO clones with pEC(50) values of 6. 14 for alpha(1a)-OCU.1-AR, 7.25 for alpha(1a)-OCU.2-AR and 7.70 for alpha(1a)-OCU.3-AR, respectively. In conclusion, the present study shows the occurrence of three splice isoforms of rabbit alpha(1a)-AR, which are unique in C-terminal sequence and in tissue distribution. They show similar pharmacological profiles in binding studies but alpha(1a)-OCU.3-AR had the highest potency of noradrenaline in functional studies in spite of the lowest receptor density. These findings suggest that the structure of C-terminus of alpha(1a)-ARs may give the characteristic functional profile.
Suzuki
F
F
Department of Pharmacology, School of Medicine, Fukui Medical University, Matsuoka, Fukui 910-1193, Japan.
Taniguchi
T
T
Takauji
R
R
Murata
S
S
Muramatsu
I
I
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000032:analysis; D015723:Gene Library; D006168:Guinea Pigs; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011869:Radioligand Assay; D018340:Receptors, Adrenergic, alpha-1 / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
C547713:ADRA1A protein, human; D018076:DNA, Complementary; D020033:Protein Isoforms; D018340:Receptors, Adrenergic, alpha-1; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium