HUMAN CALCITONIN RECEPTOR IS DIRECTLY TARGETED TO AND RETAINED IN THEBASOLATERAL SURFACE OF MDCK CELLS

The human calcitonin receptor (hCTR) is expressed in polarized cells o f the kidney, bone, and nervous system. In the kidney, hCTRs are found in cells of the distal nephron to which blood-borne calcitonin has ac cess only at the basolateral surface. We expressed hCTR subtypes 1 and 2 in Madin-Darby canine kidney (MDCK) cells to establish a cell model useful for delineating the molecular mechanisms underlying hCTR polar ity. Selective cell surface incubation demonstrated functional polarit y of hCTRs by equilibrium binding or cross-linking of radioiodinated s almon calcitonin (I-125-sCT) and cAMP accumulation stimulated by sCT. We estimated that at the steady state there are 40-fold more hCTRs on the basolateral than on the apical side. Domain-selective cell surface biotinylation followed by immunoblotting of streptavidin-agarose-frac tionated biotinylated glycoproteins independently confirmed the polari zed distribution of FLAG epitope-tagged hCTR-2 in the basolateral doma in. Confocal microscopy of immunostained receptors revealed that hCTRs are concentrated on a lateral subdomain of the basolateral membrane. Cell surface arrival assay of newly formed receptors demonstrated that direct delivery to the basolateral domain is the mechanism by which h CTRs become polarized. Measurement of receptor turnover on the basolat eral surface showed that retention contributes to hCTR distribution at the steady state.