Apical membrane and intracellular distribution of endogenous alpha 2A-adrenergic receptors in MDCK cells.
The purpose of the current studies was to characterize the endogenous alpha 2-adrenergic receptor (AR) subtypes present in Madin-Darby canine kidney (MDCK) cells and to determine their level of expression and pattern of distribution. By saturation binding analysis with [3H]MK-912, MDCK cells expressed high levels of alpha 2-ARs with a maximum receptor density (Bmax) of 798 +/- 55 fmol/mg protein and an equilibrium dissociation constant (Kd) of 0.98 +/- 0.32 nM. Competitive binding studies using prazosin, oxymetazoline, phentolamine, and epinephrine to displace [3H]MK-912 demonstrated inhibition constant (Ki) values of 1,270 +/- 250, 5.0 +/- 0.4, 5.5 +/- 0.3, and 392 +/- 150 nM (n = 3), respectively. In Northern blot analysis we found that MDCK cells expressed transcripts encoding alpha 2A-AR and not alpha 2B-AR or alpha 2C-AR. Surface binding experiments suggested that approximately 60% of alpha 2A-ARs are distributed at the cell surface domain. Specific binding of [3H]MK-912 to soluble apical and basolateral surface proteins isolated by surface biotinylation indicated the expression of surface alpha 2A-ARs was limited to the apical domain of MDCK cells. No alpha 2A-ARs were detected on the basolateral surface. We conclude that endogenous alpha 2A-ARs are targeted to the apical domain of MDCK cells and that the intracellular compartment may contain ARs as a reservoir for de novo cell surface expression or, alternatively, may represent internalized receptors.
Okusa
M D
MD
Department of Medicine, University of Virginia Health Sciences Center, Charlottesville 22908.
Lynch
K R
KR
Rosin
D L
DL
Huang
L
L
Wei
Y Y
YY
D000317:Adrenergic alpha-Antagonists / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D007425:Intracellular Membranes / Q000378:metabolism*; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D011807:Quinolizines / Q000378:metabolism; D011942:Receptors, Adrenergic, alpha / Q000145:classification / Q000378:metabolism*; D014018:Tissue Distribution
D000317:Adrenergic alpha-Antagonists; D011807:Quinolizines; D011942:Receptors, Adrenergic, alpha; C054290:L 657743