Insect Biochem. Vol. 19, No. 6, pp. 573-579, 1989 0020-1790/89$3.00+ 0.00 Printed in Great Britain. All rights reserved Copyright © 1989PergamonPress plc A PEPTIDE CONTAINING THE CELL ADHESION SEQUENCE RGD CAN MEDIATE DEGRANULATION AND CELL ADHESION OF CRAYFISH GRANULAR HAEMOCYTES IN VITRO MATS W. JOHANSSON and KENNETH SODERH,~LL Department of Physiological Botany, University of Uppsala, Box 540, S-751 21 Uppsala, Sweden (Received 2 January 1989; revised and accepted 7 April 1989) Al~tract--The synthetic peptide Gly-Arg-Gly-Asp-Ser (GRGDS) (which corresponds to a fragment of fibronectin and contains its cell adhesion sequence RGD) caused degranulation and spreading of monolayers of isolated granular haemocytes of the crayfish Pacifastacus leniusculus in vitro. When coated on glass coverslips, this RGD-containing peptide could mediate cell attachment of granular cells in vitro. A control peptide, Gly-Arg-Gly-Glu-Ser (GRGES), did not have these activities. Thus, GRGDS imitates the biological activities in vitro of the cell adhesion factor recently purified from crayfish haemocytes. This suggests that the sequence Arg-Gly-Asp (RGD), which is responsible for the cell adhesion activities of a number of vertebrate proteins, may also be involved in degranulation and cell adhesion of arthropod haemocytes. This is the first report describing direct activities by an RGD-containing peptide towards invertebrate cells in vitro, and the first indication of the presence of an RGD-recognizing receptor on invertebrate haemocytes. Key Word Index: Pacifastacus leniusculus, degranulation, exocytosis, cell adhesion, Arg-Gly-Asp, RGD, fibronectin INTRODUCTION From haemocyte lysate of the crayfish Pacifastacus leniusculus, a cell adhesion factor (CAF) has recently been purified and characterized (Johansson and S6derh/ili, 1988). When coated on glass coverslips, this protein can mediate the attachment and spread- ing of crayfish haemocytes in vitro (Johansson and S6derh/ill, 1988); and if added to monolayers of granular haemocytes on glass coverslips, it causes degranulation (Johansson and Sfderh~ill, 1989). It has a molecular mass of 76 kDa (Johansson and S6derh/iil, 1988) and an isoelectric point of 7.2 (Jo- hansson and S6derh/ill, 1989). The protein has been immunolocalized to the granules of the crayfish semigranular and the granular haemocytes (Beauvais, Johansson, Latg6 and S6derh/ill, unpublished), wherefrom it can be released during exocytosis (degranulation) in vitro. However, the biological activities of the secreted cell adhesion factor are not generated until the prophenoloxidase (proPO) activating system (for review, see S6derh~ill 1982; Ashida et al., 1982; Sfderh~ill and Smith 1986), which is similarly secreted by exocytosis (Johansson and S6derh/ill, 1985), is activated (Johansson and S6derhfill, 1988). For a number of vertebrate cell adhesion proteins, including fibronectin, it has been established that the Address all correspondence and reprint requests to: Kenneth S6derhfill, Department of Physiological Botany, University of Uppsala, Box 540, S-751 21 Uppsala, Sweden. short peptide sequence Arg-Gly-Asp (RGD) is crucial for their cell adhesion activities (for a recent review, see Ruoslahti and Pierschbacher, 1987). Thus, pep- tides containing this sequence can imitate the activity of intact cell adhesion proteins in vitro, substrata coated with the peptides themselves can mediate cell adhesion of several vertebrate cell types (see e.g. Pierschbacher and Ruoslahti, 1984a, b). Furthermore, RGD-containing peptides in solution can competitively inhibit cell adhesion to a substratum of e.g. fibronectin, by binding to the RGD-recognizing cell adhesion receptor of the cell (Pierschbacher and Ruoslahti 1984a, b; Yamada and Kennedy, 1984; Akiyama and Yamada, 1985; Hayman et al., 1985). Small changes in the RGD sequence, such as the replacement of the aspartic acid residue by glutamic acid, eliminate the activity of the peptide (Pierschbacher and Ruoslahti, 1984b). Some of these cell adhesion receptors are specific in the sense that they bind to the RGD sequence of only one intact cell adhesion protein, but all recognize small RGD-containing peptides (Ruoslahti and Pierschbacher, 1987). The present study was undertaken to investigate whether an RGD-containing peptide also can imitate the in vitro activities of the cell adhesion factor isolated from crayfish haemocytes. This was indeed found to be the case, since the peptide Gly-Arg-Gly-Asp-Ser (GRGDS) could mediate both degranulation and cell adhesion of crayfish granular haemocytes, implying the possible existence of an RGD-recognizing receptor on arthropod haemocytes. 573