Am. J. Respir. Cell Mol. Biol., Vol 11, No. 1, Jul 1994, 7-15.
Ultrastructural localization of variant forms of cystic fibrosis transmembrane conductance regulator in human bronchial epithelial of xenografts
Y Yang, JF Engelhardt and JM Wilson
Department of Internal Medicine, University of Michigan Medical School, Ann Arbor.
Cystic fibrosis (CF) is caused by mutations in the gene encoding a cyclic
adenosine monophosphate (cAMP)-regulated chloride (CI) channel called the
CF transmembrane conductance regulator (CFTR). Previous in vitro studies
have indicated that the most common mutation, delta F508 CFTR (a deletion
of phenylalanine 508), encodes a protein that is trapped in the endoplasmic
reticulum (ER), leading to loss of cAMP- regulated CI transport at the
plasma membrane. Another common variant, G551D CFTR (a G-->D missense
mutation at position 551), is properly transported to the plasma membrane
but is unresponsive to cAMP. These hypotheses are based primarily on
studies in culture cells. We have attempted to extend the in vitro
experiments by characterizing the molecular pathogenesis of the common
mutations, delta F508 and G551D, in the context of a more relevant setting,
the pseudostratified epithelium of a proximal human airway. Recombinant
adenoviruses were used to transduce normal and variant forms of CFTR into
surface epithelial cells of human bronchial xenografts grown in nu/nu mice.
Recombinant forms of CFTR RNA and protein were expressed at levels that
exceed expression of the endogenous gene. Immunolocalization of CFTR at the
light and electron microscopic level indicated that products of the wild
type and G551D alleles are found primarily at the apical plasma membrane of
ciliated cells, while the delta F508 variant is distributed diffusely
throughout the ER. Our data support previous observations primarily made in
vitro that the G551D variant is a dysfunctional channel that is properly
processed and that the delta F508 variant undergoes biosynthetic arrest at
the level of the ER.
