title: Human calcitonin receptor is directly targeted to and retained in the basolateral surface of MDCK cells.
abstract: The human calcitonin receptor (hCTR) is expressed in polarized cells of the kidney, bone, and nervous system. In the kidney, hCTRs are found in cells of the distal nephron to which blood-borne calcitonin has access 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 polarity. Selective cell surface incubation demonstrated functional polarity of hCTRs by equilibrium binding or cross-linking of radioiodinated salmon calcitonin (125I-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-fractionated biotinylated glycoproteins independently confirmed the polarized distribution of FLAG epitope-tagged hCTR-2 in the basolateral domain. 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 hCTRs become polarized. Measurement of receptor turnover on the basolateral surface showed that retention contributes to hCTR distribution at the steady state.
authors.affiliation: Division of Molecular Medicine, Department of Medicine, Cornell University Medical College, New York, New York 10021, USA.
authors.forename: D R
authors.initials: DR
authors.lastname: Nussenzveig
authors.forename: M D
authors.initials: MD
authors.lastname: Matos
authors.forename: C N
authors.initials: CN
authors.lastname: Thaw
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002116:Calcitonin / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology* / Q000648:ultrastructure; D002463:Cell Membrane Permeability / Q000502:physiology; D016764:Cell Polarity / Q000502:physiology*; D004285:Dogs; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010455:Peptides; D021382:Protein Sorting Signals / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D018003:Receptors, Calcitonin / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D014162:Transfection
chemical_list: D009842:Oligopeptides; D010455:Peptides; D021382:Protein Sorting Signals; D018003:Receptors, Calcitonin; D011994:Recombinant Proteins; D002116:Calcitonin; C085706:FLAG peptide