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Cell surface-collagen interaction : Binding sites for type I collagen in human and bovine fibroblasts
Cell Biology International Reports, Vol. 10, No. 3, March 1986
160
CELL SURFACE-COLLAGEN INTERACTION : BINDING SITES FOR TYPE I COLLAGEN IN HUMAN AND BOVINE FIBROBLASTS
O. Selmin, G. Bressan °, M. Bortolussi & D. Volpin ° I n s t i t u t e of Human Anatomy and I n s t i t u t e of Histology and Embryology° , University of Padova, I t a l y Recent studies have shown the influence of the e x t r a c e l l u l a r matrix on d i f f e r e n t c e l l u l a r functions l i k e adhesion, d i f f e r e n t i a t i o n and p r o l i f e r a t i o n 3. To examine the i n t e r a c t i o n of collagen with the cell membrane, we have studied the binding of radioiodinated human type I collagen (1251-collagen) to human dermal f i b r o b l a s t s and bovine f e t a l ligamentum nuchae f i b r o b l a s t s ( 110 days of age). The binding of 1251-collagen to both types of f i b r o b l a s t s in monolayer reached the e q u i l i b r i u m in 2-3 hours at 26°C and appeared to be a saturable process with a Kd of approximately I0-8M. The number of collagen binding sites was 0.5 x I 0 6 / c e l i in human f i b r o b l a s t s , while i t was about 10 times lower in ligamentum nuchae fibroblasts.
Similar results have been reported f o r the binding of 1251-1abelled
type I r a t collagen to mouse f i b r o b l a s t s in monolayer1'2 The binding c h a r a c t e r i s t i c s of 1251-collagen to suspensions of p u r i f i e d membranes of the same c e l l s were s i m i l a r to those obtained with f i b r o b l a s t s in monolayer. The d i s s o c i a t i o n of 1251-collagen from the membranes was increased by the addition of an excess of unlabelled type I collagen, while i t was unaffected by equimolar concentrations of f i b r o n e c t i n . In an attempt to i d e n t i f y possible membrane proteins responsible f o r the i n t e r a c t i o n with 1251-collagen, membrane preparations of both types of f i b r o b l a s t s were s o l u b i l i z e d with Nonidet P-4O and the extracts obtained were subjected to affinity
chromatography on a column of agarose-type I collagen. The material
eluted from the a f f i n i t y
column was radioiodinated and analyzed by sodium
dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography. The results have shown a few bands ranging from 15 to 100 Kd; one of these bands (33 Kd) was s p e c i f i c a l l y enriched in membrane preparations from human f i b r o b l a s t s which had a high number of binding sites f o r 1251-collagen. The present results suggest the existence of membrane receptors f o r type I collagen in human and bovine f i b r o b l a s t s . REFERENCES I. Goldberg, B. (1979) Cell 16, 265-275. 2. Goldberg, B. (1982) J. Cell B i o l . 95, 747-751. 3. Kleinman, H.K., Klebe, R.J., Martin, G.R. (1983) J. Cell Biol. 88, 473-485.
0309-1651/86/030160-01/$03.00/0
@ 1986 Academic Press Inc. (London) Ltd,
160
CELL SURFACE-COLLAGEN INTERACTION : BINDING SITES FOR TYPE I COLLAGEN IN HUMAN AND BOVINE FIBROBLASTS
O. Selmin, G. Bressan °, M. Bortolussi & D. Volpin ° I n s t i t u t e of Human Anatomy and I n s t i t u t e of Histology and Embryology° , University of Padova, I t a l y Recent studies have shown the influence of the e x t r a c e l l u l a r matrix on d i f f e r e n t c e l l u l a r functions l i k e adhesion, d i f f e r e n t i a t i o n and p r o l i f e r a t i o n 3. To examine the i n t e r a c t i o n of collagen with the cell membrane, we have studied the binding of radioiodinated human type I collagen (1251-collagen) to human dermal f i b r o b l a s t s and bovine f e t a l ligamentum nuchae f i b r o b l a s t s ( 110 days of age). The binding of 1251-collagen to both types of f i b r o b l a s t s in monolayer reached the e q u i l i b r i u m in 2-3 hours at 26°C and appeared to be a saturable process with a Kd of approximately I0-8M. The number of collagen binding sites was 0.5 x I 0 6 / c e l i in human f i b r o b l a s t s , while i t was about 10 times lower in ligamentum nuchae fibroblasts.
Similar results have been reported f o r the binding of 1251-1abelled
type I r a t collagen to mouse f i b r o b l a s t s in monolayer1'2 The binding c h a r a c t e r i s t i c s of 1251-collagen to suspensions of p u r i f i e d membranes of the same c e l l s were s i m i l a r to those obtained with f i b r o b l a s t s in monolayer. The d i s s o c i a t i o n of 1251-collagen from the membranes was increased by the addition of an excess of unlabelled type I collagen, while i t was unaffected by equimolar concentrations of f i b r o n e c t i n . In an attempt to i d e n t i f y possible membrane proteins responsible f o r the i n t e r a c t i o n with 1251-collagen, membrane preparations of both types of f i b r o b l a s t s were s o l u b i l i z e d with Nonidet P-4O and the extracts obtained were subjected to affinity
chromatography on a column of agarose-type I collagen. The material
eluted from the a f f i n i t y
column was radioiodinated and analyzed by sodium
dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography. The results have shown a few bands ranging from 15 to 100 Kd; one of these bands (33 Kd) was s p e c i f i c a l l y enriched in membrane preparations from human f i b r o b l a s t s which had a high number of binding sites f o r 1251-collagen. The present results suggest the existence of membrane receptors f o r type I collagen in human and bovine f i b r o b l a s t s . REFERENCES I. Goldberg, B. (1979) Cell 16, 265-275. 2. Goldberg, B. (1982) J. Cell B i o l . 95, 747-751. 3. Kleinman, H.K., Klebe, R.J., Martin, G.R. (1983) J. Cell Biol. 88, 473-485.
0309-1651/86/030160-01/$03.00/0
@ 1986 Academic Press Inc. (London) Ltd,