![Figure 1. The atypical SH3 domain-binding motif of SLP-65 allows constitutive association with Grb2. (A) Amino acid sequence comparison (single-letter code) of human (top) and chicken (bottom) SLP-65 proteins. The human SLP-65 Δexon 8-splice variant lacks 23 amino acids in the central proline-rich region encompassing an atypical SH3 domain-binding motif P-(x)3-R-(x)2-K-P (x can be any amino acid). This particular motif is missing in the 9 amino acid–shortened chicken SLP-65 variant used in this study (chicken s-SLP-65). The sequence of a synthetic peptide, SLP-65[pep], which represents the atypical SH3-domain binding motif and which was used for affinity purifications is indicated. (B) Affinity purification of human SLP-65 and SLP-65 Δexon 8 by individual Grb2 domains. Human Ramos B cells were left untreated (lanes 1, 3, and 5) or stimulated through their BCR (lanes 2, 4, and 6) and cleared cellular lysates were subjected to affinity purifications with GST fusion proteins harboring isolated Grb2 domains (N-terminal SH3 domain, lanes 1-2; central SH2 domain, lanes 3-4; C-terminal SH3 domain, lanes 5-6). Purified proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and SLP-65 isoforms were detected by immunoblotting with anti–SLP-65 antibodies. The relative molecular mass of marker protein is indicated on the left. (C) Binding parameters of the constitutive SLP-65/Grb2 complex. Recombinantly produced SLP-65 was mixed stepwise in an isothermal titration calorimeter with either full-length Grb2 (left panel) or the isolated N- and C-terminal Grb2 SH3 domains (middle and right panels, respectively). Resulting changes of the heating power (top panel each) on association between binding partners were integrated and plotted versus the molar ratio Grb2/SLP-65 in the bottom panels. Curves according to a single-site binding model were fitted to obtain the indicated dissociation constants (Kd). NBD indicates no binding detectable. (D) Affinity purification of Grb2 by the SLP-65 peptide. grb2–/– DT40 cells were reconstituted with either wild-type Grb2 (lanes 1-2) or a mutant version carrying an inactivated C-terminal SH3 domain (W193K, lanes 3-4). From the cleared lysates of these cells, Grb2 was precipitated using an immobilized biotinylated SLP-65 peptide (SLP-65[pep], described in panel A) and visualized by anti-Grb2 immunoblotting. The relative molecular mass of marker protein is indicated on the left.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/108/12/10.1182_blood-2006-02-005397/4/zh80230604480001.jpeg?Expires=1722779079&Signature=F5iCAo2Fm1C9UID1oyskZy1VWhUBn6ND1KEf30qgfs8Ws6mncNHpMEFIG6sNnqvHkVTv3UMWQy0Y1BC6MYpxOPYCAd2j-OOVwlBmS7O8B3R3bF8dQWsHJ2SRJS2kX6zj7ylbGbfeLNp5flORoMX1QnY26A7JnEEJOJoT3sfJRdQJlaBtqZZKxz0as2H~a8P0i8AdXGb6~9YZuqxErdjemdmEC327kzjkaRLo-kVJcZm7H6iQDf0j27osZSefIn091iO5ZUFuHGmNG17d~PTja5zFqQMZstXix4kTeZdoLToMYGe5uNN2gqptueLgEjOhq7dOcWxPG8c8yC~o7z5Rbw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The atypical SH3 domain-binding motif of SLP-65 allows constitutive association with Grb2. (A) Amino acid sequence comparison (single-letter code) of human (top) and chicken (bottom) SLP-65 proteins. The human SLP-65 Δexon 8-splice variant lacks 23 amino acids in the central proline-rich region encompassing an atypical SH3 domain-binding motif P-(x)3-R-(x)2-K-P (x can be any amino acid). This particular motif is missing in the 9 amino acid–shortened chicken SLP-65 variant used in this study (chicken s-SLP-65). The sequence of a synthetic peptide, SLP-65[pep], which represents the atypical SH3-domain binding motif and which was used for affinity purifications is indicated. (B) Affinity purification of human SLP-65 and SLP-65 Δexon 8 by individual Grb2 domains. Human Ramos B cells were left untreated (lanes 1, 3, and 5) or stimulated through their BCR (lanes 2, 4, and 6) and cleared cellular lysates were subjected to affinity purifications with GST fusion proteins harboring isolated Grb2 domains (N-terminal SH3 domain, lanes 1-2; central SH2 domain, lanes 3-4; C-terminal SH3 domain, lanes 5-6). Purified proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and SLP-65 isoforms were detected by immunoblotting with anti–SLP-65 antibodies. The relative molecular mass of marker protein is indicated on the left. (C) Binding parameters of the constitutive SLP-65/Grb2 complex. Recombinantly produced SLP-65 was mixed stepwise in an isothermal titration calorimeter with either full-length Grb2 (left panel) or the isolated N- and C-terminal Grb2 SH3 domains (middle and right panels, respectively). Resulting changes of the heating power (top panel each) on association between binding partners were integrated and plotted versus the molar ratio Grb2/SLP-65 in the bottom panels. Curves according to a single-site binding model were fitted to obtain the indicated dissociation constants (Kd). NBD indicates no binding detectable. (D) Affinity purification of Grb2 by the SLP-65 peptide. grb2–/– DT40 cells were reconstituted with either wild-type Grb2 (lanes 1-2) or a mutant version carrying an inactivated C-terminal SH3 domain (W193K, lanes 3-4). From the cleared lysates of these cells, Grb2 was precipitated using an immobilized biotinylated SLP-65 peptide (SLP-65[pep], described in panel A) and visualized by anti-Grb2 immunoblotting. The relative molecular mass of marker protein is indicated on the left.
