Increase in the expression of a family of small guanosine triphosphate-binding proteins, rab proteins, during induced phagocyte differentiation.

Abstract

Rab is a newly identified family of small G-proteins that share 35-70% homology with the yeast Sec4p and Yptlp involved in the regulation of the secretory pathway. Mature phagocytes display functions requiring organized intracellular traffic and, for this reason, we questioned whether phagocyte differentiation could correlate with the increased expression of rab proteins. Rabbit antisera raised against the recombinant proteins rablAp, 2p, 4p, and 6p were able to detect the corresponding proteins in the human monoblast leukemic cell line U937. When these cells were induced to differentiate into monocyte/ macrophage-like cells displaying functional characteristics of a normal phagocyte, rablAp, 2p, 4p, and 6p were increased and this correlated with an increase in the rab transcripts. Using a rab5 probe, we also observed an increased expression of the rab5 gene in differentiated cells. Similarly, differentiation of the human leukemic myeloblast HL60 cell line along either monocyte or granulocyte pathways induced an increased expression of the rab proteins. Rab proteins were also detected in human neutrophils and in guinea pig alveolar macrophages. As degranulation is one of the phagocyte functions acquired in the late stage of differentiation, we investigated whether rab proteins would be involved in this process. Although rab proteins were tightly membrane bound, none of them was detected in the specific or azurophil granules purified from human neutrophils. The increased expression of rab proteins in mature phagocytes suggests that they may promote functions highly developed in these cells. (J. Clin. Invest. 1991. 87:901-907.)

Subcellular Distribution and Differential Expression of Endogenous ADP-ribosylation Factor 6 in Mammalian Cells

“…The differentiation was determined by their adhesion to substratum and a substantial increase in the expression of total small GTP-binding proteins (Fig. 6B, lower panel), as previously reported (16). When equal amounts of total proteins prepared from undifferentiated and differentiated HL-60 cells were used for immunoblotting by the anti-ARF6 monoclonal antibody (3F8.1), the levels of ARF6 unexpectedly decreased in differentiated HL-60 cells (Fig.…”

“…Maturation of HL-60 cells was induced as described by MaridonneauParini et al (16). Briefly, HL-60 cells were cultured at a density of 2 ϫ 10 5 cells/ml and exposed to 20 ng/ml phorbol 12-myristate 13-acetate for 4 days.…”

The small GTP-binding protein rab4 is associated with early endosomes

“…The purified antiserum was specific for rab4p ( Fig. la) and did not recognize purified, recombinant proteins corresponding to rabl, -2, -3, -5, or -6 (38).…”

The src-family protein-tyrosine kinase p59hck is located on the secretory granules in human neutrophils and translocates towards the phagosome during cell activation

“…For immunolocalization of Hck in HL60 cells, the cells were differentiated with 20 ng/ml phorbol myristate acetate for 3 days on glass coverslips [32]. In some experiments, neutrophils or HL60 cells were stimulated with 4.5 mg/ml zymosan particles that had been opsonized in human serum [31].…”

“…For immunoblotting, proteins were separated by SDS/PAGE (8 % gels), transferred to nitrocellulose and blotted as previously described [32]. Histocompatibility locus antigen (HLA) class I (plasma membrane), myeloperoxidase (azurophil granules) and lactoferrin (specific granules) were assayed by ELISA with antibodies kindly donated by N. Borregaard [28,35] (State University Hospital, Copenhagen, Denmark).…”