The objective of this work is to determine the mechanism by which reticulocytes and earlier red cells, remodel their plasma membranes during the maturation to erythrocytes. Mature red cells from different species do not retain precisely the same functions found in their respective reticulocytes. (That is, loss of function is species specific). Little is known about the cellular processing and targeting for this selective loss. The long-term objective is to establish the mechanism by which the selected proteins, such as the transferrin receptor or nucleoside transporter, are recognized and removed from the plasma membrane. Since the nucleoside transporter is retained in some species, are there differences between lost and retained transporters? Since the externalization process can occur after new mRNA formation has ceased, these events are either programmed much earlier and are selectively activated or the regulatory """"""""factors"""""""", such as heme, accumulate in the cell till a critical concentration is reached. An avian cell line, infected with a temperature sensitive, erythroblastic virus will be employed to study the early events in differentiation, under controlled conditions, to determine when transferrin receptor (and other functions) begin to be lost from the cell. The methods employed to detect these functions (and others) are ongoing in the laboratory. The experiments will examine the levels of total Hb synthesis and transferrin receptor (TFR) synthesis before and after induction. Induction requires a change to 42 degrees C plus the addition of hemin or anemic chicken serum. The fate of the TFR and protein synthesis are then followed over a period of time until the majority of the cells have matured (i.e., no further mRNA synthesis). The fate of a number of recognizable membrane proteins is also followed after release into the incubation medium. A short-term objective of the project is to assess the origin of the truncated, circulating transferrin receptor in the plasma, i.e., cleaved directly from the cell surface or from released exosomes. This new information may lead to improved assays for detection of clinical anemias and improved discrimination between effects of iron deficiency and marrow dysfunction in anemia as well as how red cells target proteins for externalization.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK040299-04A1
Application #
3240470
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1988-09-26
Project End
1996-01-31
Budget Start
1993-02-01
Budget End
1994-01-31
Support Year
4
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Mcgill University
Department
Type
DUNS #
City
Montreal
State
PQ
Country
Canada
Zip Code
H3 0-G4
Weeks 2nd, James L; Zoraghi, Roya; Francis, Sharron H et al. (2007) N-Terminal domain of phosphodiesterase-11A4 (PDE11A4) decreases affinity of the catalytic site for substrates and tadalafil, and is involved in oligomerization. Biochemistry 46:10353-64
Zoraghi, Roya; Francis, Sharron H; Corbin, Jackie D (2007) Critical amino acids in phosphodiesterase-5 catalytic site that provide for high-affinity interaction with cyclic guanosine monophosphate and inhibitors. Biochemistry 46:13554-63
Blount, Mitsi A; Zoraghi, Roya; Bessay, Emmanuel P et al. (2007) Conversion of phosphodiesterase-5 (PDE5) catalytic site to higher affinity by PDE5 inhibitors. J Pharmacol Exp Ther 323:730-7
Zoraghi, Roya; Corbin, Jackie D; Francis, Sharron H (2006) Phosphodiesterase-5 Gln817 is critical for cGMP, vardenafil, or sildenafil affinity: its orientation impacts cGMP but not cAMP affinity. J Biol Chem 281:5553-8
Blount, Mitsi A; Zoraghi, Roya; Ke, Hengming et al. (2006) A 46-amino acid segment in phosphodiesterase-5 GAF-B domain provides for high vardenafil potency over sildenafil and tadalafil and is involved in phosphodiesterase-5 dimerization. Mol Pharmacol 70:1822-31
Corbin, J; Francis, S; Zoraghi, R (2006) Tyrosine-612 in PDE5 contributes to higher affinity for vardenafil over sildenafil. Int J Impot Res 18:251-7
Zoraghi, Roya; Bessay, Emmanuel P; Corbin, Jackie D et al. (2005) Structural and functional features in human PDE5A1 regulatory domain that provide for allosteric cGMP binding, dimerization, and regulation. J Biol Chem 280:12051-63
Zoraghi, Roya; Corbin, Jackie D; Francis, Sharron H (2004) Properties and functions of GAF domains in cyclic nucleotide phosphodiesterases and other proteins. Mol Pharmacol 65:267-78
Johnstone, R M; Mathew, A; Setchenska, M S et al. (1998) Loss of glucose transport in developing avian red cells. Eur J Cell Biol 75:66-77
Johnstone, R M (1996) Cleavage of the transferrin receptor by human granulocytes: differential proteolysis of the exosome-bound TFR. J Cell Physiol 168:333-45

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