PMID- 10347183
OWN - NLM
STAT- MEDLINE
DCOM- 19990701
LR  - 20190508
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 274
IP  - 23
DP  - 1999 Jun 4
TI  - Topological and functional analysis of the human reduced folate carrier by
      hemagglutinin epitope insertion.
PG  - 16269-78
AB  - The membrane topology of the human reduced folate carrier protein (591 amino
      acids) was assessed by single insertions of the hemagglutinin epitope into nine
      sites of the protein. Reduced folate carrier-deficient Chinese hamster ovary
      cells expressing each of these constructs were probed with anti-hemagglutinin
      epitope monoclonal antibodies to assess whether the insertion was exposed to the 
      external environment or to the cytoplasm. The results are consistent with the
      12-transmembrane topology predicted for this protein. The hemagglutinin epitope
      insertion mutants were also tested for their effects on the function of the
      reduced folate carrier. For these studies, each of the constructs had a
      carboxyl-terminal fusion of the enhanced green fluorescent protein to monitor and
      quantitate expression. Insertions into the external loop between transmembrane
      regions 7 and 8 (Pro-297), the cytoplasmic loop between transmembrane regions 6
      and 7 (Ser-225), and near the cytoplasmic amino and carboxyl termini (Pro-20 and 
      Gly-492, respectively) had minor effects on methotrexate binding and uptake. The 
      insertion into the cytoplasmic loop between transmembrane regions 10 and 11
      (Gln-385) greatly reduced both binding and uptake of methotrexate, whereas the
      insertion into the external loop between transmembrane regions 11 and 12
      (Pro-427) selectively interfered with uptake but not binding.
FAU - Ferguson, P L
AU  - Ferguson PL
AD  - Department of Microbiology and Immunology, University of Western Ontario, London,
      Ontario N6A 5C1, Canada.
FAU - Flintoff, W F
AU  - Flintoff WF
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (Antibodies, Monoclonal)
RN  - 0 (Carrier Proteins)
RN  - 0 (DNA Primers)
RN  - 0 (Epitopes)
RN  - 0 (Hemagglutinin Glycoproteins, Influenza Virus)
RN  - 0 (Membrane Proteins)
RN  - 0 (Membrane Transport Proteins)
RN  - 0 (Reduced Folate Carrier Protein)
RN  - 0 (SLC19A1 protein, human)
RN  - 0 (SLC19A2 protein, human)
RN  - 935E97BOY8 (Folic Acid)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - Antibodies, Monoclonal/immunology
MH  - Base Sequence
MH  - Blotting, Western
MH  - CHO Cells
MH  - Carrier Proteins/*chemistry
MH  - Cell Membrane/*chemistry
MH  - Cricetinae
MH  - DNA Primers
MH  - *Epitopes
MH  - Folic Acid/*chemistry
MH  - Glycosylation
MH  - Hemagglutinin Glycoproteins, Influenza Virus/*chemistry/immunology
MH  - Humans
MH  - *Membrane Proteins
MH  - *Membrane Transport Proteins
MH  - Microscopy, Fluorescence
MH  - Molecular Sequence Data
MH  - Mutagenesis
MH  - Protein Conformation
MH  - Protein Structure, Secondary
MH  - Reduced Folate Carrier Protein
MH  - Structure-Activity Relationship
MH  - Transfection
EDAT- 1999/05/29 00:00
MHDA- 1999/05/29 00:01
CRDT- 1999/05/29 00:00
PHST- 1999/05/29 00:00 [pubmed]
PHST- 1999/05/29 00:01 [medline]
PHST- 1999/05/29 00:00 [entrez]
AID - 10.1074/jbc.274.23.16269 [doi]
PST - ppublish
SO  - J Biol Chem. 1999 Jun 4;274(23):16269-78. doi: 10.1074/jbc.274.23.16269.