Cellular internalization of proinsulin C-peptide.
Proinsulin C-peptide is known to bind specifically to cell membranes and to exert intracellular effects, but whether it is internalized in target cells is unknown. In this study, using confocal microscopy and immunostained or rhodamine-labeled peptide, we show that C-peptide is internalized and localized to the cytosol of Swiss 3T3 and HEK-293 cells. In addition, transport into nuclei was found using the labeled peptide. The internalization was followed at 37 degrees C for up to 1 h, and was reduced at 4 degrees C and after preincubation with pertussis toxin. Hence, it is concluded to occur via an energy-dependent, pertussis toxin-sensitive mechanism and without detectable degradation within the experimental time course. Surface plasmon resonance measurements demonstrated binding of HEK-293 cell extract components to C-peptide, and subsequent elution of bound material revealed the components to be intracellular proteins. The identification of C-peptide cellular internalization, intracellular binding proteins, absence of rapid subsequent C-peptide degradation and apparent nuclear internalization support a maintained activity similar to that of an intracrine peptide hormone. Hence, the data suggest the possibility of one further C-peptide site of action.
Lindahl
E
E
Department of Medical Biochemistry and Biophysics (MBB), Karolinska Institutet, 171 77, Stockholm, Sweden.
Nyman
U
U
Melles
E
E
Sigmundsson
K
K
Ståhlberg
M
M
Wahren
J
J
Obrink
B
B
Shafqat
J
J
Joseph
B
B
Jörnvall
H
H
D016475:3T3 Cells; D000818:Animals; D002096:C-Peptide / Q000378:metabolism*; D002457:Cell Extracts; D004705:Endocytosis / Q000187:drug effects; D005434:Flow Cytometry; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D009862:Online Systems; D037342:Pertussis Toxin / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D012235:Rhodamines / Q000378:metabolism; D013696:Temperature
D002096:C-Peptide; D002457:Cell Extracts; D012235:Rhodamines; D037342:Pertussis Toxin