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Heterogeneity of laminin binding sites in M2R mouse melanoma cells
HUMAN LAMININ B1 CHAIN: A MULTIDOMAIN PROTEIN WITH GENE (LAMB1) LOCUS IN THE q22 REGION OF CHROMOSOME 7.
HETEROGENEITY OF LAMININ BINDING SITES IN M=R MOUSE MELANOMA CELLS. M.V. Marquezini 2 S. Sonohara~ R. Chammas and M.M. Brentani. Lab. Oncologia Experimental da Faculdade de Medicina da Universidade de Sao Paulo, Brasil. M2R melanoma cells were grown in Dulbeeco's medium containing 10% FBS until semi confluence. Specific sigmoidal binding curves were obtained by a whole cell method in monolayer culture, using 1251-1aminin, SA 5,2 UCi/]~ (0,2 nM-6 nM) in the absence and presence of 200-fold excess of laminin or PI laminin fragment. In the presence of 0,2% albumin, Statchard and Hill analysis suggests the existence of at least two binding components. The first one corresponds to the linear part of the Scatchard plot, presenting a Kd of about 1,0 nM and a maximum number of binding sites (Bo) of 138.000 sites/cell. The second one correspond~2~o the curvilinear part of the graph. Binding of l-laminin to this first component is inhibited only by unlabelled laminin, or its PI fragment (97%) whereas binding to the second component can be also inhibited by albumin (60%) and fibronectin (72%). On assays in the absence of 0~2% albumin, Scatchard analysis reveals vaulted ceiling plots, indicating that the second component may have multiple binding sites (as its Hill coefficient is about 2.0) which display positive cooperativity. Based on the statistical mechanical model, the binding parameters of this second component were: Kd of about 16,1 nM~ Bo=1.420.000 sites/cell and eooperativity positive parameter of 27 to 29-fold. Supported by FAPESP (84/1923-0).
T. Pikkarainen a, R. Eddyb, Y. Fukushimab, M. Byers b, T. Shows b, T. Pih/ajaniemt c, M. Saraste d and K. Tryggvason a" Departments of Biochemistry a and Medical Biochemistry c, University of Oulu, Oulu, Finland, Department of Human Genetics°, Roswall Park Memorial Institute, Buffalo, NY, USA and Department of Medical Chemistry d, University of Halsinki, Helsinki, Finland. Laminin is a basement membrane specific protein that has been shown to play a role for the adhesion, migration, mitoganssis and differentiation of cells. It interacts with other basement membrane components such as type IV collagen, proteoglycan and antactin. The molecule is composed of three polypeptida chains (A, B1 and B2). In the present work we have isolated several cDNA clones that provide the first and nearly complete (>93%) amino acid sequence of the human laminin B1 chain. The clones covered 5103 nucleotides of which 4965 were in an open reading frame encoding 1655 amino acids from the C-terminal and of the polypsptide chain. Two types of internal homology repeats were found in clusters within the polypeptida. The type A repeats contained about 50 amino acids of which 8 were cystaine. These repeats were present in two clusters that were separated by about 200 amino acids. There were two copies of consecutive type B repeats of about 40 amino acids. Computer analysis of the amino acid sequence of the B1 chain revealed the presence of 8 structurally distinct domains that contained cystsine-rich repeats, globular regions and helical structures. The human laminin B1 gene (LAMB1) was located to the q22 region on chromosome 7 using somatic cell hybrid and in situ hybridization techniques.
6? EXTRACELLULAR THE F O R M A T I O N
68 M A T R I X (ECM) C O M P O N E N T S D U R I N G OF M E S O D E R M IN RAT E M B R Y O S
IMMUNOLOCALIZATION OF FIB~ONECTINS fiN) AND LArIININ (LN) IN THI PI~ EMBRYO, V, RICHOLIXU,2), T, DARRIbER[ (I) JC. BOUCAUT (I),~I, E. FLECHVN {2J and JF' THIERY i3), (I) blologle Experlmentale, UA 11.35 IJmverslte I-, et M Curie. 75~30 I:'aris Cedex ~ ~ (2) INRA, 78350 Jouv en Josas ; (75) Instltut d~.rnbrvoloqle,CNRf., College de France, 94130 Noqent sur Marne, France
R.E. P o e l m a n n , M.M.T. M e n t i n k and N. G i r a r d Dept. o f A n a t o m y , L e i d e n U n i v e r s i t y , The N e t h e r l a n d s and L a b . d ' I m m u n o c h i m i e , Centre H e n r i B e c q u e r e l , Rouen, France. The f o r m a t i o n of the m e s o d e r m c o m p a r t m e n t in rat e m b r y o s is a c o m p l e x p r o c e s s , b e c a u s e s e v e r a l s o u r c e s g i v e r i s e to v a r i o u s cell types. The m o s t i m p o r t a n t are the p r i m i t i v e s t r e a k and the n e u r a l crest, t h o u g h a l s o a n u m b e r o f p l a c o d e s g i v e r i s e to m e s o d e r m cells. The E C M p l a y s an i m p o r t a n t r o l e d u r ing the d i f f e r e n t i a t i o n f r o m e p i b l a s t and e c t o d e r m into m e s o d e r m and m e s e c t o d e r m . The d i s t r i b u t i o n of f i b r o n e c t i n and h y a l u r o n a t e is s t u d i e d in p r e s o m i t e - 2 0 s o m i t e stages. Rat e m b r y o s , c u l t u r e d in vitro, are t r e a t e d w i t h h y a l u r o n i d a s e , to d e s t r o y the ECM. The f o r m a t i o n of the m e s o d e r m and m e s e c t o d e r m is i n v e s t i g a t e d w i t h a c e l l l a b e l i n g t e c h n i q u e b a s e d on l e c t i n - c o l l o i d a l g o l d t r e a t m e n t of the p r i m i t i v e s t r e a k and the n e u r a l crest. The p r e s e n c e of b a s a l l a m i n a c o n s t i t uents, e.g. l a m i n i n and c o l l a g e n IV, is ana l y s e d w i t h a n t i b o d i e s to i n v e s t i g a t e the s i t e s o f o r i g i n of l a b e l e d c e l l s in the e p i b l a s t and the e c t o d e r m . T h e s e c o m b i n e d t e c h n i q u e s p r e s e n t b o t h a f a t e - m a p of the m e s o d e r m in e a r l y rat e m b r y o s and a tool for the m e c h a n i s m of the m e s o d e r m d i f f e r e n t i a t i o n .
The ontogenv and the distrlbuLlonof flbronectms ffN~ and larnlnm (LNJ were studied m the Piq embr v0 by indirect immunofluorescence using antl-porcme FN and antl-murlne LN antibodiesrespecbvelv ExtracelIular EN first appear m the early blastocvst before the mtqraUon 01 endodermal cells FN are then located in the expanded blastocvst, at the Interfac~ trophectoderm-extraembryomc endoderm Mesodermal ceils migrate m a trlclimensmnal network of fibrillar FN FN are also m the extraembryonK membranes ~chorlon and yolk sac wail) contiguous to FN-rlch basement membranes underlying embryomc ectoderm and mesoderm LN apbears later than FN, it is located between the trophectoderm and the extraembryanlc endoderm which produces it. LN also accumulates at the basal surface of the embryonic ectoderm at the onset 0t gastrulat~on. In the extraembrvon~c membranes, LN secreted bv mesodermal cells is located at the interface endoderm-mesoderm and trophectoderm-mesoderm. These results suggest that FN allow the migration 0t enoodermai anO mesodermal cells by providing them w~th a suitable subst.rate.Once locahzed, these cells synthesize LN. probably In order to stabilize their interactions with the underlyingextracellularmaterial and epltheha 23S
HETEROGENEITY OF LAMININ BINDING SITES IN M=R MOUSE MELANOMA CELLS. M.V. Marquezini 2 S. Sonohara~ R. Chammas and M.M. Brentani. Lab. Oncologia Experimental da Faculdade de Medicina da Universidade de Sao Paulo, Brasil. M2R melanoma cells were grown in Dulbeeco's medium containing 10% FBS until semi confluence. Specific sigmoidal binding curves were obtained by a whole cell method in monolayer culture, using 1251-1aminin, SA 5,2 UCi/]~ (0,2 nM-6 nM) in the absence and presence of 200-fold excess of laminin or PI laminin fragment. In the presence of 0,2% albumin, Statchard and Hill analysis suggests the existence of at least two binding components. The first one corresponds to the linear part of the Scatchard plot, presenting a Kd of about 1,0 nM and a maximum number of binding sites (Bo) of 138.000 sites/cell. The second one correspond~2~o the curvilinear part of the graph. Binding of l-laminin to this first component is inhibited only by unlabelled laminin, or its PI fragment (97%) whereas binding to the second component can be also inhibited by albumin (60%) and fibronectin (72%). On assays in the absence of 0~2% albumin, Scatchard analysis reveals vaulted ceiling plots, indicating that the second component may have multiple binding sites (as its Hill coefficient is about 2.0) which display positive cooperativity. Based on the statistical mechanical model, the binding parameters of this second component were: Kd of about 16,1 nM~ Bo=1.420.000 sites/cell and eooperativity positive parameter of 27 to 29-fold. Supported by FAPESP (84/1923-0).
T. Pikkarainen a, R. Eddyb, Y. Fukushimab, M. Byers b, T. Shows b, T. Pih/ajaniemt c, M. Saraste d and K. Tryggvason a" Departments of Biochemistry a and Medical Biochemistry c, University of Oulu, Oulu, Finland, Department of Human Genetics°, Roswall Park Memorial Institute, Buffalo, NY, USA and Department of Medical Chemistry d, University of Halsinki, Helsinki, Finland. Laminin is a basement membrane specific protein that has been shown to play a role for the adhesion, migration, mitoganssis and differentiation of cells. It interacts with other basement membrane components such as type IV collagen, proteoglycan and antactin. The molecule is composed of three polypeptida chains (A, B1 and B2). In the present work we have isolated several cDNA clones that provide the first and nearly complete (>93%) amino acid sequence of the human laminin B1 chain. The clones covered 5103 nucleotides of which 4965 were in an open reading frame encoding 1655 amino acids from the C-terminal and of the polypsptide chain. Two types of internal homology repeats were found in clusters within the polypeptida. The type A repeats contained about 50 amino acids of which 8 were cystaine. These repeats were present in two clusters that were separated by about 200 amino acids. There were two copies of consecutive type B repeats of about 40 amino acids. Computer analysis of the amino acid sequence of the B1 chain revealed the presence of 8 structurally distinct domains that contained cystsine-rich repeats, globular regions and helical structures. The human laminin B1 gene (LAMB1) was located to the q22 region on chromosome 7 using somatic cell hybrid and in situ hybridization techniques.
6? EXTRACELLULAR THE F O R M A T I O N
68 M A T R I X (ECM) C O M P O N E N T S D U R I N G OF M E S O D E R M IN RAT E M B R Y O S
IMMUNOLOCALIZATION OF FIB~ONECTINS fiN) AND LArIININ (LN) IN THI PI~ EMBRYO, V, RICHOLIXU,2), T, DARRIbER[ (I) JC. BOUCAUT (I),~I, E. FLECHVN {2J and JF' THIERY i3), (I) blologle Experlmentale, UA 11.35 IJmverslte I-, et M Curie. 75~30 I:'aris Cedex ~ ~ (2) INRA, 78350 Jouv en Josas ; (75) Instltut d~.rnbrvoloqle,CNRf., College de France, 94130 Noqent sur Marne, France
R.E. P o e l m a n n , M.M.T. M e n t i n k and N. G i r a r d Dept. o f A n a t o m y , L e i d e n U n i v e r s i t y , The N e t h e r l a n d s and L a b . d ' I m m u n o c h i m i e , Centre H e n r i B e c q u e r e l , Rouen, France. The f o r m a t i o n of the m e s o d e r m c o m p a r t m e n t in rat e m b r y o s is a c o m p l e x p r o c e s s , b e c a u s e s e v e r a l s o u r c e s g i v e r i s e to v a r i o u s cell types. The m o s t i m p o r t a n t are the p r i m i t i v e s t r e a k and the n e u r a l crest, t h o u g h a l s o a n u m b e r o f p l a c o d e s g i v e r i s e to m e s o d e r m cells. The E C M p l a y s an i m p o r t a n t r o l e d u r ing the d i f f e r e n t i a t i o n f r o m e p i b l a s t and e c t o d e r m into m e s o d e r m and m e s e c t o d e r m . The d i s t r i b u t i o n of f i b r o n e c t i n and h y a l u r o n a t e is s t u d i e d in p r e s o m i t e - 2 0 s o m i t e stages. Rat e m b r y o s , c u l t u r e d in vitro, are t r e a t e d w i t h h y a l u r o n i d a s e , to d e s t r o y the ECM. The f o r m a t i o n of the m e s o d e r m and m e s e c t o d e r m is i n v e s t i g a t e d w i t h a c e l l l a b e l i n g t e c h n i q u e b a s e d on l e c t i n - c o l l o i d a l g o l d t r e a t m e n t of the p r i m i t i v e s t r e a k and the n e u r a l crest. The p r e s e n c e of b a s a l l a m i n a c o n s t i t uents, e.g. l a m i n i n and c o l l a g e n IV, is ana l y s e d w i t h a n t i b o d i e s to i n v e s t i g a t e the s i t e s o f o r i g i n of l a b e l e d c e l l s in the e p i b l a s t and the e c t o d e r m . T h e s e c o m b i n e d t e c h n i q u e s p r e s e n t b o t h a f a t e - m a p of the m e s o d e r m in e a r l y rat e m b r y o s and a tool for the m e c h a n i s m of the m e s o d e r m d i f f e r e n t i a t i o n .
The ontogenv and the distrlbuLlonof flbronectms ffN~ and larnlnm (LNJ were studied m the Piq embr v0 by indirect immunofluorescence using antl-porcme FN and antl-murlne LN antibodiesrespecbvelv ExtracelIular EN first appear m the early blastocvst before the mtqraUon 01 endodermal cells FN are then located in the expanded blastocvst, at the Interfac~ trophectoderm-extraembryomc endoderm Mesodermal ceils migrate m a trlclimensmnal network of fibrillar FN FN are also m the extraembryonK membranes ~chorlon and yolk sac wail) contiguous to FN-rlch basement membranes underlying embryomc ectoderm and mesoderm LN apbears later than FN, it is located between the trophectoderm and the extraembryanlc endoderm which produces it. LN also accumulates at the basal surface of the embryonic ectoderm at the onset 0t gastrulat~on. In the extraembrvon~c membranes, LN secreted bv mesodermal cells is located at the interface endoderm-mesoderm and trophectoderm-mesoderm. These results suggest that FN allow the migration 0t enoodermai anO mesodermal cells by providing them w~th a suitable subst.rate.Once locahzed, these cells synthesize LN. probably In order to stabilize their interactions with the underlyingextracellularmaterial and epltheha 23S