Integrins are a family of proteins known to mediate attachment of cells to extracellular matrix materials. The substratum specificity and cation dependence of specific integrin heterodimers have been extensively characterized, and to a lesser degree specialized roles in cell attachment versus dendricity have been defined in some cell types. In the past decade, melanocyte attachment rate and morphology have been found to have strong substratum dependence, suggesting a major role for integrins in these processes. In order to investigate this aspect of pigment cell biology, human newborn melanocytes were subjected to flow cytometry analysis and plated on a variety of substrata under conditions known to promote or block the binding of specific integrin pairs. Melanocyte attachment to laminin and type IV collagen was promoted by Mg2+ and Mn2+ but not by Ca2+, in the range of concentrations examined. However, dendrite outgrowth from melanocytes already attached on laminin or type IV collagen was promoted by Ca2+ to a far greater degree than by Mg2+, and Mn2+ had no effect on dendrite outgrowth. Flow cytometry analysis revealed that melanocytes expressed beta 1, alpha 2, alpha 3, alpha 5, alpha 6 and alpha v integrin subunits as well as the alpha v beta 3 heterodimer. The influence of substratum on the profile of integrin expression was minimal, but alpha 6 and beta 1 integrins were observed by confocal microscopy to be expressed over the entire cell surface, while alpha 2, alpha 5 and alpha v beta 3 integrins localized along dendritic processes or at their tips. In accordance with the implications of these distribution patterns, anti-beta 1 and anti-alpha 6 integrin monoclonal antibodies blocked melanocyte attachment to laminin, while anti-alpha 2, anti-alpha 5 and anti-alpha v beta 3 inhibited dendrite outgrowth but did not block substratum attachment on either laminin or type IV collagen. On the basis of these data and the known characteristics of integrin molecules, we conclude that melanocyte attachment to laminin is mediated primarily by alpha 6 beta 1 integrin in a Ca(2+)-independent, Mg(2+)- and/or Mn(2+)-dependent manner, while dendrite outgrowth on laminin and type IV collagen requires extracellular Ca2+ and is mediated by alpha v beta 3 as well as alpha 2 and alpha 5 integrins.

REFERENCES

Adams
J. C.
,
Watt
F. M.
(
1989
).
Fibronectin inhibits the terminal differentiation of human keratinocytes.
Nature
340
,
307
309
Akers
R. M.
,
Mosher
D. F.
,
Lilien
J. E.
(
1981
).
Promotion of retinal neurite outgrowth by substratum-bounded fibronectin.
Dev. Biol
86
,
179
188
Albelda
S. M.
,
Buck
C. A.
(
1990
).
Integrins and other cell adhesion molecules.
FASEB J
4
,
2868
2880
Aznavoorian
S.
,
Stracke
M. L.
,
Krutzsch
H.
,
Schiffmann
E.
,
Liotta
L. A.
(
1990
).
Signal transduction for chemotaxis and hepatotaxis by matrix molecules in tumor cells.
J. Cell Biol
110
,
1427
1438
Blum
J. L.
,
Zeigler
M. E.
,
Wicha
M. S.
(
1987
).
Regulation of rat mammary gene expression by extracellular matrix components.
Exp. Cell Res
173
,
322
340
Bozyczko
D.
,
Horwitz
A. F.
(
1986
).
The participation of a putative cell surface receptor for laminin and fibronectin in peripheral neurite extension.
J. Neurosci
6
,
1241
1251
Calof
A. L.
,
Lander
A. D.
(
1991
).
Relationship between neuronal migration and cell-substratum adhesion: laminin and merosin promote olfactory neuronal migration but are anti-adhesive.
J. Cell Biol
115
,
779
794
Clyman
R. I.
,
Mauray
F.
,
Kramer
R. H.
(
1992
).
1and 3integrinshave different roles in the adhesion and migration of vascular smooth muscle cells on extracellular matrix.
Exp. Cell Res
200
,
272
284
DiPersio
C. M.
,
Jackson
D. A.
,
Zaret
K. S.
(
1991
).
The extracellular matrix coordinately modulates liver transcription factors and hepatocyte morphology.
Mol. Cell. Biol
11
,
4405
4414
D'Souza
S. E.
,
Ginsberg
M. H.
,
Matsueda
G. R.
,
Plow
E. F.
(
1991
).
A discrete sequence in a platelet integrin is involved in ligand recognition.
Nature
350
,
66
68
Elices
M. J.
,
Hemler
M. E.
(
1989
).
The human integrin VLA-2 is a collagen receptor on some cells and a collagen/laminin receptor on others.
Proc. Nat. Acad. Sci. USA
86
,
9906
9910
Elices
M. J.
,
Urry
L. A.
,
Hemler
M. E.
(
1991
).
Receptor functions for the integrin VLA-3: fibronectin, collagen and laminin binding are differentially influenced by ARG-GLY-ASP peptide and by divalent cations.
J. Cell Biol
112
,
169
181
Forsberg
E.
,
Paulsson
M.
,
Timpl
R.
,
Johansson
S.
(
1990
).
Characterization of a laminin receptor on rat hepatocytes.
J. Biol. Chem
265
,
6376
6381
Gailit
J.
,
Ruoslahti
E.
(
1988
).
Regulation of the fibronectin receptor affinity by divalent cations.
J. Biol. Chem
263
,
12927
12932
Gilchrest
B. A.
,
Vrabel
M. A.
,
Flynn
E.
,
Szabo
G.
(
1984
).
Selective cultivation of human melanocytes from newborn and adult epidermis.
J. Invest. Dermatol
83
,
370
376
Gilchrest
B. A.
,
Albert
L. S.
,
Karassik
R. L.
,
Yaar
M.
(
1985
).
Substrate influences human epidermal melanocyte attachment and spreading in vitro.
In Vitro
21
,
114
119
Gulino
D.
,
Boudignon
C.
,
Zhang
L.
,
Concord
E.
,
Rabiet
M.-J.
,
Marguerie
G.
(
1992
).
Ca2+-binding properties of the platelet glycoprotein IIb ligand-interacting domain.
J. Biol. Chem
267
,
1001
1007
Hadley
M. E.
,
Quevedo
W. C.
(
1966
).
Vertebrate epidermal melanin unit.
Nature
209
,
1334
1335
Hall
D. E.
,
Reichardt
L. F.
,
Crowley
E.
,
Holley
B.
,
Moezzi
H.
,
Sonnenberg
A.
,
Damsky
C. H.
(
1990
).
The/ 1and6/ 1integrin heterodimers mediate cell attachment to distinct sites on laminin.
J. Cell Biol
110
,
2175
2184
Heidemann
S. R.
,
Joshi
H. C.
,
Schechter
A.
,
Fletcher
J. R.
,
Bothwell
M.
(
1985
).
Synergistic effects of cyclic AMP and nerve growth factor on neurite outgrowth and microtubule stability of PC12 cells.
J. Cell Biol
100
,
916
927
Hynes
R. O.
(
1992
).
Integrins: versatility, modulation, and signaling in cell adhesion.
Cell
69
,
11
25
Hynes
R. O.
,
Lander
A. D.
(
1992
).
Contact and adhesive specificities in the associations, migrations and targeting of cells and axons.
Cell
68
,
303
322
Kirchhofer
D.
,
Grzesiak
J.
,
Pierschbacher
M. D.
(
1991
).
Calcium as a potential physiological regulator of integrin-mediated cell adhesion.
J. Biol. Chem
266
,
4471
4477
Klebe
R. J.
,
Hall
J. R.
,
Rosenberger
P.
,
Dickey
W. D.
(
1977
).
Cell attachment to collagen: the ionic requirements.
Exp. Cell Res
110
,
419
425
Kramer
R. H.
,
Cheng
Y.-F.
,
Clyman
R.
(
1990
).
Human microvascularendothelial cells use 1and3integrin receptor complexes to attach to laminin.
J. Cell Biol
111
,
1233
1243
Lallier
T.
,
Bronner-Fraser
M.
(
1992
).
1integrin on neural crest cells recognizes some laminin substrata in a Ca2+-independent manner.
J. Cell Biol
119
,
1335
1345
Lander
A. D.
,
Fujii
D. K.
,
Reichardt
L. F.
(
1985
).
Laminin is associated with the neurite outgrowth-promoting factors found in conditioned media.
Proc. Nat. Acad. Sci. USA
82
,
2183
2187
Leavesley
D. I.
,
Schwartz
M. A.
,
Rosenfeld
M.
,
Cheresh
D. A.
(
1993
).
Integrin1-and 3-mediated endothelial cell migration is triggered through distinct signaling mechanisms.
J. Cell Biol
121
,
163
170
Loftus
J. C.
,
Plow
E. F.
,
O'Toole
T. E.
,
Glass
A.
,
Felinger
A. L.
,
Ginsterg
M. H.
(
1990
).
A3integrin mutation abolishes ligand binding and alters divalent cation-dependent conformation.
Science
249
,
915
918
Manthorpe
M.
,
Engvall
E.
,
Ruoslahti
E.
,
Longo
F. M.
,
Davis
G. E.
,
Varon
S.
(
1983
).
Laminin promotes neuritic regeneration from cultured peripheral and central neurons.
J. Cell Biol
97
,
1882
1890
Masumoto
A.
,
Hemler
M. E.
(
1993
).
Mutation of putative divalent cation sites in the4subunit of the integrin VLA-4: distinct effects on adhesion to CS1/fibronectin, VCAM-1, and invasin.
J. Cell Biol
123
,
245
253
Menon
G. K.
,
Grayson
S.
,
Elias
P. M.
(
1985
).
Ionic calcium reservoirs in mammalian epidermis: ultrastructural localization by ion-capture cytochemistry.
J. Invest. Dermatol
84
,
508
512
Morelli
J. G.
,
Yohn
J. J.
,
Zekman
T.
,
Norris
D. A.
(
1993
).
Melanocyte movement in vitro: role of matrix proteins and integrin receptors.
J. Invest. Dermatol
101
,
605
608
Naeyaert
J. M.
,
Eller
M.
,
Gordon
P. R.
,
Park
H.-Y.
,
Gilchrest
B. A.
(
1991
).
Pigment content of cultured human melanocytes does not correlate with tyrosinase message level.
Br. J. Dermatol
125
,
297
303
Neugebauer
K. M.
,
Emmett
C. L.
,
Venstrom
K. A.
,
Reichardt
L. F.
(
1991
).
Vitronectin and thrombospondin promote retinal neurite outgrowth: developmental regulation and role of integrins.
Neuron
6
,
345
358
O'Shea
K. S.
,
Liu
L.-H. J.
,
Dixit
V. M.
(
1991
).
Thrombospondin and a 140 kd fragment promote adhesion and neurite outgrowth from embryonic central and peripheral neurons and from PC12 cells.
Neuron
7
,
231
237
Park
H.-Y.
,
Russakovsky
V.
,
Ohno
S.
,
Gilchrest
B. A.
(
1993
).
Theisoform of protein kinase C stimulates human melanogenesis by activating tyrosinase in pigment cells.
J. Biol. Chem
268
,
11742
11749
Ranson
M.
,
Posen
S.
,
Mason
R. S.
(
1988
).
Extracellular matrix modulates the function of human melanocytes but not melanoma cells.
J. Cell. Physiol
136
,
281
288
Rogers
S. L.
,
Letourneau
P. C.
,
Palm
S. L.
,
McCarthy
J.
,
Furcht
L. T.
(
1983
).
Neurite extension by peripheral and central nervous system neurons in response to substratum-bound fibronectin and laminin.
Dev. Biol
98
,
212
220
Scott
G.
,
Ryan
D. H.
,
McCarthy
J. B.
(
1992
).
Molecular mechanisms of human melanocyte attachment to fibronectin.
J. Invest. Dermatol
99
,
787
794
Sonnenberg
A.
,
Modderman
P. W.
,
Hogervorst
F.
(
1988
).
Laminin receptor on platelets is the integrin VLA-6.
Nature
336
,
487
489
Sonnenberg
A.
,
Linders
C. J. T.
,
Modderman
P. W.
,
Damsky
C. H.
,
Aumailley
M.
,
Timpl
R.
(
1990
).
Integrin recognition of different cell-binding fragments of laminin (P1, E3, E8) and evidence that6 1but not6 4functions as a major receptor for fragment E8.
J. Cell Biol
110
,
2145
2155
Staatz
W. D.
,
Rajpara
S. M.
,
Wayner
E. A.
,
Carter
W. G.
,
Santoro
S. A.
(
1989
).
The membrane glycoprotein Ia-IIa (VLA-2) complex mediates the Mg2+-dependent adhesion of platelets to collagen.
J. Cell Biol
108
,
1917
1924
Streuli
C. H.
,
Bailey
N.
,
Bissell
M. J.
(
1991
).
Control of mammary epithelial differentiation: basement membrane induces tissue-specific gene expression in the absence of cell-cell interaction and morphological polarity.
J. Cell Biol
115
,
1383
1395
Svoboda
K. K. H.
(
1992
).
Intracellular localization of types I and II collagen mRNA and endoplasmic reticulum in embryonic corneal epithelia.
J. Cell Sci
100
,
23
33
Takeichi
M.
(
1977
).
Functional correlation between cell adhesive properties and some cell surface proteins.
J. Cell Biol
75
,
464
474
Tawil
N.
,
Wilson
P.
,
Carbonetto
S.
(
1993
).
Integrins in point contacts mediate cell spreading: factors that regulate integrin accumulation in point contacts vs. focal contacts.
J Cell Biol
120
,
261
271
Tomasek
J. J.
,
Hay
E. D.
,
Fujiwara
K.
(
1982
).
Collagen modulates cell shape and cytoskeleton of embryonic corneal and fibroma fibroblasts: distribution of actin,-actinin, and myosin.
Dev. Biol
92
,
107
122
Tomaselli
K. J.
,
Hall
D. E.
,
Flier
L. A.
,
Gehlsen
K. R.
,
Turner
D. C.
,
Carbonetto
S.
,
Reichardt
L. F.
(
1990
).
A neuronal cell line (PC12) expresses two1-class integrins- 1and3 1- that recognize different neurite outgrowth-promoting domains in laminin.
Neuron
5
,
651
662
Turner
D. C.
,
Flier
L. A.
,
Carbonetto
S.
(
1987
).
Magnesium-dependent attachment and neurite outgrowth by PC12 cells on collagen and laminin substrata.
Dev. Biol
121
,
510
525
Watt
F. M.
,
Kubler
M.-D.
,
Hotchin
N. A.
,
Nicholson
L. J.
,
Adams
J. C.
(
1993
).
Regulation of keratinocyte terminal differentiation by integrin-extracellular matrix interactions.
J. Cell Sci
106
,
175
182
Woodley
D. T.
,
Kimberly
C. W.
,
O'Keefe
E. J.
(
1990
).
Type IV collagen and fibronectin enhance human keratinocyte thymidine incorporation and spreading in the absence of soluble growth factors.
J. Invest. Dermatol
94
,
139
143
Yaar
M.
,
Woodley
D. T.
,
Gilchrest
B. A.
(
1988
).
Human nevo-cellular nevus cells are surrounded by basement membrane components.
Lab. Invest
58
,
157
162
Yaar
M.
,
Gilchrest
B. A.
(
1991
).
Human melanocyte growth and differentiation: a decade of new data.
J. Invest. Dermatol
97
,
611
617
Zambruno
G.
,
Marchisio
P. C.
,
Melchiori
A.
,
Bondanza
S.
,
Cancedda
R.
,
De Luca
M.
(
1993
).
Expression of integrin receptors and their role in adhesion, spreading and migration of normal human melanocytes.
J. Cell Sci
105
,
179
190
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