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.1997 Aug 5;94(16):8569-74.
doi: 10.1073/pnas.94.16.8569.

The SH3p4/Sh3p8/SH3p13 protein family: binding partners for synaptojanin and dynamin via a Grb2-like Src homology 3 domain

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The SH3p4/Sh3p8/SH3p13 protein family: binding partners for synaptojanin and dynamin via a Grb2-like Src homology 3 domain

N Ringstad et al. Proc Natl Acad Sci U S A..

Abstract

The GTPase dynamin I and the inositol 5-phosphatase synaptojanin are nerve terminal proteins implicated in synaptic vesicle recycling. Both proteins contain COOH-terminal proline-rich domains that can interact with a variety of Src homology 3 (SH3) domains. A major physiological binding partner for dynamin I and synaptojanin in the nervous system is amphiphysin I, an SH3 domain-containing protein also concentrated in nerve terminals. We have used the proline-rich tail of synaptojanin to screen a rat brain library by the two-hybrid method to identify additional interacting partners of synaptojanin. Three related proteins containing SH3 domains that are closely related to the SH3 domains of Grb2 were isolated: SH3p4, SH3p8, and SH3p13. Further biochemical studies demonstrated that the SH3p4/8/13 proteins bind to both synaptojanin and dynamin I. The SH3p4/8/13 transcripts are differentially expressed in tissues: SH3p4 mRNA was detected only in brain, SH3p13 mRNA was present in brain and testis, and the SH3p8 transcript was detected at similar levels in multiple tissues. Members of the SH3p4/8/13 protein family were found to be concentrated in nerve terminals, and pools of synaptojanin and dynamin I were coprecipitated from brain extracts with antibodies recognizing SH3p4/8/13. These findings underscore the important role of SH3-mediated interactions in synaptic vesicle recycling.

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Figures

Figure 1
Figure 1
Structure of the rat SH3p4, SH3p8, and SH3p13 proteins isolated by the two-hybrid screen. (A) Domain cartoon of the cDNAs isolated by the screen. Dashed boxes represent portions of the ORF that were not contained in the isolated cDNAs and subsequently were cloned by PCR. Thin lines represent untranslated regions. Numbers in parentheses refer to the number of clones isolated for each cDNA by the two-hybrid screen. (B) Sequence alignment of SH3p4, SH3p8, and SH3p13 proteins. The complete amino acid sequences of the three proteins was aligned by the clustal method using the Megalign module of the Lasergene package (DNAstar, Madison, WI). Regions of identity between the three proteins are outlined by black boxes. Note the high similarity among the three proteins, which is interrupted only by a highly variable amino acid stretch that separates the NH2-terminal coiled-coil domain portion from the COOH-terminal SH3 domain.
Figure 2
Figure 2
Overlay assays of a total brain homogenate. (A) GST fusion proteins comprising either the COOH-terminal fragments of the SH3p4/8/13 proteins (including both the variable and the SH3 domains) or the SH3 domain of amphiphysin I were used to probe blots of a total brain homogenate by overlay (far Western blotting). (B) The same homogenate was probed by Western blotting with antibodies directed against synaptojanin and dynamin. Bands corresponding to synaptojanin and dynamin are indicated by arrowheads. The figure demonstrates the specific and direct interaction of the SH3p4/8/13 proteins with synaptojanin and dynamin I.
Figure 3
Figure 3
Affinity purification of a brain extract onto immobilized SH3 domains of the SH3p4 and SH3p13 proteins. (A) Protein staining of the starting material (lane 1), flow through (lane 2), bound material (lane 3), and GST fusion protein alone (lane 4). An excess of control fusion proteins was loaded on the control lane. (B) Western blot analysis of material affinity-purified from the brain extract with the SH3 domains of the SH3p4 and SH3p13 proteins or with GST alone, demonstrating that the affinity-purified bands of 100 and 150 kDa correspond to dynamin I and synaptojanin, respectively.
Figure 4
Figure 4
Northern blot analysis of the pattern of expression of SH3p4/8/13 mRNAs in different tissues. Oligonucleotide probes derived from the cDNAs of SH3p4, SH3p8, and SH3p13 were used to probe a multiple tissue blot. Quantity and integrity of mRNA present on the filters was verified by probing for glyceraldehyde-3-phosphate dehydrogenase transcripts (data not shown).
Figure 5
Figure 5
Western blot analysis of SH3p4/8/13 proteins in brain extract. (A) Polyclonal rabbit antibodies raised against the COOH-terminal regions of SH3p4 (serum 1) and SH3p13 (serum 2), respectively were affinity-purified and used by Western blotting to label His-tagged COOH-terminal regions of the three SH3p proteins corresponding to the immunogens used to raise antisera. Serum 1 recognizes primarily SH3p13 whereas serum 2 recognizes intensely all three proteins. (B) The same antibodies were used to blot a total homogenate of rat brain. Serum 2 labels two bands, whereas serum 1 labels only the lower band. The electrophoretic motility of the two bands, 40 and 45 kDa, respectively, is in good agreement with the molecular masses of the SH3p4/8/13 proteins.
Figure 6
Figure 6
Colocalization of SH3p4/8/13 immunoreactivity with dynamin I, synaptojanin, and amphiphysin I in nerve terminals in rat brain. Indirect immunofluorescence of frozen sections of rat brain stem using serum 1 and serum 2 and mAbs directed against dynamin I, synaptojanin, and amphiphysin I. In all cases immunoreactivity is represented by a punctate nerve terminal pattern, superimposed on a diffuse cytosolic staining. Slight differences between staining patterns (Upper) can be accounted for by variability within the neurons in the region of the brain examined.
Figure 7
Figure 7
Colocalization of SH3p4/8/13 immunoreactivity with the synaptic vesicle marker synaptophysin in cultured hippocampal neurons. Two-week-old cultures of hippocampal neurons were immunostained with serum 1 (A) and serum 2 (B) and counterstained with the anti-synaptophysin mAb C7.2 (C andD). SH3p4/8/13 and synaptophysin immunoreactivities colocalize in presynaptic nerve terminals visible as puncta surrounding the perikarya and processes of the cultured neurons.
Figure 8
Figure 8
Coimmunoprecipitation of dynamin and synaptojanin with SH3p4/8/13 proteins. Material immunoprecipitated using anti-SH3p4/8/13 antibodies (serum 2) from a brain extract was processed for Western blotting with antibodies directed against synaptojanin, dynamin, the SH3p4/8/13 proteins (serum 1), and, as a control, with antibodies directed against the nerve terminal protein glutamic acid decarboxylase (GAD). Starting material (SM), supernatant (S), and pellet (P) fractions are shown. Anti-dynamin and synaptojanin Western blots were processed using an alkaline phosphatase detection system while anti-glutamic acid decarboxylase and anti-SH3p4/8/13 immunoreactivity was detected by ECL.
Figure 9
Figure 9
The SH3 domains of the SH3p4/8/13 proteins are closely related to the SH3 domains of Grb2. SH3 domains of various proteins were aligned using the clustal method and displayed as a phylogenetic tree.
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