Identification of cysteine-rich epidermal growth factor-like domain 1alpha (CRELD1alpha) as a novel alpha1A-adrenoceptor-down-regulating protein and establishment of an alpha1L-adrenoceptor-expressing cell line.
Two distinct alpha(1)-adrenoceptor phenotypes (alpha(1A)- and alpha(1L)-ARs) are known to originate from a single ADRA1A(alpha(1a)) gene by an as-yet-unknown mechanism. We hypothesized that an alpha(1a)-AR-interacting protein could generate the alpha(1L)-AR phenotype and we sought to identify such a protein and to examine its effects on the expression of alpha(1A) and alpha(1L) phenotypes. Cysteine-rich epidermal growth factor-like domain 1alpha (CRELD1alpha) was first identified using a yeast two-hybrid approach as an alpha(1a)-AR-interacting protein. Transfection of alpha(1a)-AR cDNA alone yielded Chinese hamster ovary (CHO) cells expressing alpha(1A)-ARs having a predominant high affinity site for prazosin, with a low proportion (<10%) of prazosin-low affinity sites (alpha(1L)-AR). Knockdown of endogenous CHO-CRELD1alpha [alpha(1a)-CKD(alpha(1A)-enhanced) cells] enhanced the expression of alpha(1A)-AR, whereas over-expression of CRELD1alpha reduced alpha(1A)-AR expression, yielding alpha(1a)-COE(alpha(1L)-dominant) cells expressing a high proportion (50%) of the alpha(1L)-AR phenotype. The ligand binding and functional agonist and antagonist profiles in alpha(1a)-CKD(alpha(1A)-enhanced) and alpha(1a)-COE(alpha(1L)-dominant) cell lines were entirely in accord with the alpha(1A)-AR and alpha(1L)-AR phenotypes observed in intact tissues. CRELD1alpha down-regulates expression of the alpha(1A)-AR, thereby enhancing the proportion of expression of the alpha(1L)-AR phenotype. The alpha(1L)-AR-expressing alpha(1a)-COE(alpha(1L)-dominant) cell line reflects accurately the phenotype of this AR observed in vivo and will facilitate development of alpha(1L)-AR-targeted drugs.
Nishimune
Atsushi
A
Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, School of Medicine, University of Fukui, Eiheiji, Fukui, Japan.
Suzuki
Fumiko
F
Yoshiki
Hatsumi
H
Morishima
Shigeru
S
Muramatsu
Ikunobu
I
D000818:Animals; D016466:CHO Cells; D015815:Cell Adhesion Molecules / Q000302:isolation & purification* / Q000494:pharmacology / Q000502:physiology*; D006224:Cricetinae; D003412:Cricetulus; D015536:Down-Regulation; D016326:Extracellular Matrix Proteins / Q000302:isolation & purification* / Q000494:pharmacology / Q000502:physiology*; D005260:Female; D018340:Receptors, Adrenergic, alpha-1 / Q000378:metabolism*
C465506:CRELD1 protein, human; D015815:Cell Adhesion Molecules; D016326:Extracellular Matrix Proteins; D018340:Receptors, Adrenergic, alpha-1