©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
A Binding Site for SH3 Domains Targets Dynamin to Coated Pits (*)

(Received for publication, October 11, 1995)

Howard S. Shpetner (§) Jonathan S. Herskovits (1) Richard B. Vallee

From the Cell Biology Group, Worcester Foundation for Biomedical Research, Shrewsbury, Massachusetts 01545 and Department of Biochemistry, University of Massachusetts Medical School, Worcester, Massachusetts 01655

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Dynamin is a GTPase that plays a critical role in the very early stages of endocytosis, regulating the scission of clathrin-coated and non-clathrin-coated pits from the plasma membrane. While the ligands through which dynamin exerts its in vivo effects are unknown, dynamin exhibits in vitro binding to several proteins containing Src homology 3 (SH3) domains, as well as to microtubules and anionic phospholipids, via a basic, proline-rich C-terminal domain. To begin to identify the in vivo binding partners of dynamin, we have examined by immunofluorescence the association of mutant and wild-type forms of dynamin with plasma membranes prepared by sonication of transiently transfected cells. Wild-type dynamin was found almost exclusively in association with clathrin-containing domains. Binding to these regions was abolished by removal of a nine-amino acid sequence within the C-terminal domain encoding a candidate SH3 domain binding site. Binding did not require clathrin and resisted extraction at both high and low ionic strength, consistent with an interaction with an SH3 domain. Surprisingly, we also find that dynamin contains multiple regions involved in binding to non-clathrin-containing domains, including a 13-amino acid sequence directly upstream of the C-terminal domain. These observations suggest that a protein containing an SH3 domain is involved in recruiting dynamin to coated pits and provide the first evidence for a biological role for SH3 domains in dynamin function.


INTRODUCTION

Dynamin is a 100-kDa GTPase initially identified as a nucleotide-dissociable protein in preparations of calf brain microtubules(1, 2, 3) . Genetic studies in Drosophila indicated that a fly protein exhibiting 68% overall identity with mammalian dynamin was the product of the shibire gene(4, 5) , previously implicated in the recycling of synaptic vesicles (6, 7, 8) and, more generally, in the scission of clathrin-coated and non-clathrin-coated pits from the plasma membrane(9, 10) . Studies of mammalian cells transfected with mutant forms of dynamin also implicated the protein in the early stages of endocytosis(11, 12) . More recently, ultrastructural analysis has localized the protein to coated pits in stably transformed HeLa cells (13) and to the stalks of clathrin-coated membrane invaginations in presynaptic nerve terminals (14) .

Neither the precise role of dynamin in coated pit function nor the ligands through which it acts are known. However, dynamin has been found to bind microtubules(1, 3, 15) , anionic phospholipids(16) , and the SH3 domains of several proteins (15, 17, 18, 19, 20) in vitro, and all three types of ligands also stimulate the steady-state dynamin GTPase, in some cases by as much as 75-fold(3) . These interactions are all mediated by a basic, proline-rich 10-kDa domain at the dynamin C terminus(15, 16) , which contains several sequences similar to sites in other proteins that mediate binding to SH3 domains(21) . Dynamin also contains a pleckstrin homology (PH) (^1)domain(22, 23) , which in other proteins has been implicated in heterotrimeric G-protein and phosphatidylinositol 4,5-biphosphate binding(24, 25) .

Thus far, no biological role has been established for these in vitro interactions. However, while microtubules are not known to play a role in the early stages of endocytosis, many SH3-containing proteins are thought to transit the early stages of clathrin-mediated endocytosis via interactions with activated receptor tyrosine kinases. To begin to identify the ligands through which dynamin acts in vivo, we have examined the binding of exogenously expressed wild-type and mutant forms of dynamin to plasma membranes in COS-7 cells. We report that dynamin contains multiple regions that target the protein to both clathrin-coated and non-clathrin-coated plasma membrane domains. While the PH domain was neither necessary nor sufficient for plasma membrane binding, binding to clathrin-coated pits depended critically on a nine-amino acid sequence encoding a potential SH3 domain binding site. These findings strongly suggest that dynamin is recruited to coated pits via an SH3-containing protein, perhaps in combination with other signal-transducing proteins.


MATERIALS AND METHODS

Construction of cDNA Plasmids

All constructs were generated in the pSVL mammalian expression vector (Pharmacia Biotech Inc.). The generation of D-1, N272, N456, N651, C663, N272/C663, S45N, and D208N has been described previously(11) . The N272/C794 construct was made utilizing unique SmaI sites in pSVL and the cDNA encoding D-1. All other deletion mutants were made by ligating polymerase chain reaction products into the N272 construct using unique restriction sites. All polymerase chain reaction products contained termination codons immediately following the sequence encoding dynamin.

Preparation and Examination of Plasma Membranes by Sonication of Transfected Cells

COS-7 cells were transfected in 100-mm dishes for 3 h using DEAE-dextran according to a standard protocol(26) . 48 h post-transfection the cells were trypsinized, washed, replated onto 18-mm polylysine-coated coverslips in 12-well dishes, and maintained first at 37 and then at 4 °C(27) . The cells were then washed and sonicated at 4 °C in 6 ml of buffer B (25 mM HEPES (pH 7.0), 25 mM KCl, 2.5 mM magnesium acetate, 0.2 mM dithiothreitol) for 1 s, using a -inch tapped horn 12 mm above the coverslip at a power setting of 2.5 (Heat Systems Ultrasonics, model W380)(28) . The membranes were washed three times with 2 ml of buffer B and either fixed immediately with 3% paraformaldehyde in buffer G (20 mM HEPES (pH 6.8), 100 mM KCl, 5 mM MgCl(2), 3 mM EGTA) at 4 °C or extracted at 23 °C for 15 min with the indicated reagent in buffer B, washed three more times in buffer B for 5 min, and fixed as above. Membranes were then processed for double-label immunofluorescence (11) using the X-22 monoclonal antibody to the clathrin heavy chain (gift of Dr. Francis Brodsky) and either the RA or R2 polyclonal antibodies to dynamin(11) . Transfection efficiencies generally ranged from 5 to 8%.

Overlap Analysis of Dynamin and Clathrin Immunofluorescence

To evaluate the extent to which a dynamin immunofluorescence pattern was overlapped by its corresponding clathrin pattern, a photocopy transparency was made of the latter, from which a same-size negative (black on white) image of the original was printed photographically. A photocopy transparency of this negative image was then laid over the dynamin pattern to determine the percent of the anti-dynamin-staining spots in a region of membrane that also exhibited anti-clathrin immunofluorescence. A minimum of 200 spots was counted for each membrane. Errors represent standard deviations for five membranes. The amount of overlap expected on a random basis (32.2 + 2.2%, n = 5) was determined by performing the same analysis using randomly chosen plasma membrane regions.


RESULTS AND DISCUSSION

Membranes prepared from cells expressing wild-type dynamin exhibited bright, punctate immunofluorescence that co-localized with clathrin (Fig. 1, A and B), as has recently been reported in stably transformed HeLa cells (13). Two forms of dynamin mutated in the GTP-binding consensus sequence elements, S45N (Fig. 1, C and D) and D208N (not shown), exhibited similar immunofluorescence patterns. Wild-type dynamin was not extracted from plasma membranes by either GTP, GDP, or GTPS at 0.5 mM (not shown).


Figure 1: Double-label immunofluorescence microscopy of plasma membranes from COS-7 cells expressing recombinant dynamin. Plasma membranes were prepared by sonication of COS-7 cells overexpressing either wild-type dynamin (A and B) or the S45N (C and D), N651 (E and F), or C663 (G and H) mutants. The membranes were fixed immediately and processed for immunofluorescence. A, C, E, and G, dynamin immunofluorescence; B, D, F, and H, clathrin heavy chain immunofluorescence. Only the C663 mutant failed to co-localize with clathrin.



To identify the region within dynamin responsible for clathrin co-localization, we prepared membranes from cells expressing several mutant polypeptides lacking the GTPase, PH, and/or C-terminal domains of dynamin (diagrammed in Fig. 4A). The N-terminal truncation mutants N272 and N456 (not shown), which lack the GTPase domain, and N651 (Fig. 1, E and F), which, in addition, lacks the PH domain, exhibited the same immunofluorescence pattern as the wild-type protein, indicating that the C-terminal 201 amino acids contain a signal for binding to coated pits.


Figure 4: A, diagrams of recombinant dynamin polypeptides. D-1 is the full-length, wild-type protein of 851 amino acids. S45N and D208N are full-length proteins containing point mutations (designated by crosses) in the first and third GTP-binding sequence elements, respectively(11) . Other proteins are truncation mutants for which the N- and/or C-terminal amino acids are shown at left. Vertical lines indicate positions of the three GTP-binding sequence elements. Constructs marked with failed to bind plasma membranes; constructs marked with + exhibited minimal co-localization with clathrin. Unmarked constructs showed a high level of clathrin co-localization. B, sequence of the 121 C-terminal amino acids of dynamin I. C-terminal boundaries of deletion constructs are indicated below. Locations of sequences expected or shown to bind SH3 domains (18, 19) are bracketed above (I-VI). Loss of high level clathrin colocalization occurs within the region denoted by a shaded bar, and loss of membrane binding occurs within the region denoted by a solid bar.



A complementary polypeptide (C663) lacking 188 amino acids from the dynamin C terminus failed to co-localize with clathrin (Fig. 1, G and H). Nevertheless, this polypeptide did bind to plasma membranes, although at sites that apparently lacked clathrin (see below). Taken together these observations indicated that the C-terminal 201 amino acids of dynamin were both necessary and sufficient for binding to coated pits, while removal of the PH domain had no effect on this behavior. Subdivision of the C663 polypeptide into overlapping smaller fragments (C247, C320, and N272/C663) abolished binding to the non-clathrin-containing sites (geq1600 membranes examined for each construct). Therefore, this latter activity required sequences from more than one region of the C663 polypeptide, which could include elements of the GTPase and/or PH domains.

To define more precisely the site in the C-terminal domain required for clathrin co-localization, we examined membranes from cells expressing the N272 polypeptide containing a series of C-terminal deletions (cf. Fig. 4). Constructs N272/C839, N272/C826 (Fig. 2, A and B), N272/C808, and N272/C794 all clearly co-localized with clathrin. Quantitative analysis indicated that 80-90% of the dynamin-positive spots were coincident with clathrin-positive spots (Fig. 2E), the two shorter constructs co-localizing to a slightly lesser degree.


Figure 2: Clathrin co-localization of C-terminal truncations of the N272 deletion mutant. A-D, double-label immunofluorescence of plasma membranes containing either the N272/C826 (A and B) or N272/C746 (C and D) mutants, showing either (A and C) anti-dynamin or (B and D) anti-clathrin staining. Inset delineated in each panel is shown below, enlarged. Arrows indicate locations of dynamin spots and corresponding locations in the clathrin patterns. Clear overlap between the clathrin and dynamin patterns can be seen in the membranes containing the N272/C826 but not the N272/C746 protein. E, analysis of overlap between dynamin and clathrin immunofluorescence patterns seen in membranes containing N272 C-terminal deletion mutants. Extent of overlap was evaluated using negative image (black on white) transparencies, as described under ``Materials and Methods.'' Errors represent standard deviations for five membranes. A minimum of 200 spots was counted for each membrane.



Constructs N272/C785, N272/C765, and N272/C746 also exhibited punctate membrane immunofluorescence. However, the immunoreactive spots did not obviously co-localize with clathrin (Fig. 2, C and D), and the extent of overlap was determined to be only 36-40% (Fig. 2E), slightly higher than the value obtained by analysis of randomly selected membranes (32%; see ``Materials and Methods''). Thus, the region between amino acids 786 and 794 was essential for clathrin colocalization. This sequence (PAVPPARPG) is closely related to sequences found in other proteins, including 3BP1 and SOS, which bind to SH3 domains (reviewed in (21) ). Moreover, a peptide spanning this region was found to inhibit dynamin/SH3 interactions(18) , and other peptides spanning this region have been found to bind both SH3 domains (18) and SH3 domain-containing proteins (19) . Thus, our data are consistent with a role for SH3-containing proteins in the recruitment of dynamin to clathrin-containing domains.

To characterize further the interaction between dynamin and clathrin-coated regions, plasma membranes were prepared from cells transfected with a cDNA construct encoding the C-terminal half of dynamin(N456) and extracted using a variety of reagents. The protein resisted extraction with 0.5 M Tris, pH 9.7 (Fig. 3, A and B), as well as 0.5 M KCl or 0.1 M NaHCO(3), pH 11.0, all of which extracted clathrin. alpha-Adaptin has also been reported to be extracted from plasma membranes at high ionic strength(29) . 0.2% Triton X-100 (Fig. 3, C and D) alone extracted neither the N456 polypeptide nor clathrin from the membranes and, in combination with 0.5 M KCl, extracted only clathrin. Thus, the interaction between dynamin and clathrin-coated plasma membrane regions did not appear to require either clathrin coat proteins or anionic phospholipids, suggesting that other plasma membrane components were involved. The N456 polypeptide also resisted extraction in low ionic strength buffers (5 mM Tris, pH 7.0, with or without 0.5 mM EDTA), in which the clathrin immunofluorescence pattern was partially attenuated (not shown).


Figure 3: Salt and detergent resistance of dynamin binding to coated pits. Membranes containing the N456 deletion mutant were prepared by sonication of transfected COS-7 cells and processed for immunofluorescence as described in Fig. 1, except that just prior to fixation the cells were extracted at 23 °C for 15 min with either 0.5 M Tris/HCl, pH 9.7 (A and B) or 0.02% Triton X-100 (C and D) in buffer B and washed three times in buffer B for 5 min. A and C, dynamin immunofluorescence; B and D, clathrin heavy chain immunofluorescence. Neither treatment altered the dynamin immunofluorescence pattern, although clathrin was extracted at high ionic strength, as previously reported (28) .



To identify the site in the N272 polypeptide required for binding to the non-clathrin-containing domains, we assayed additional C-terminal truncation mutants for plasma membrane binding (cf.Fig. 4B). Neither the N272/C733 nor the N272/C693 mutants exhibited punctate plasma membrane staining (geq4000 membranes examined from multiple experiments for each construct), although they were clearly detected in intact cells. Thus, the sequence spanning amino acids 733-746 was necessary for binding to the non-clathrin-containing sites.

The role of these sites in dynamin function is unclear. However, recent studies in our laboratory have indicated that removal of the sequence between amino acids 733 and 746 also abolishes the inhibition of transferrin uptake seen in cells transfected with some mutant forms of dynamin. Moreover, the proline-rich C terminus, which contains the targeting signal for coated pits, is not required for this phenotype (30) . These observations suggest that the non-clathrin-containing domains play a role in endocytosis of transferrin. Conceivably, these domains could contain components destined for the coated pit, since wild-type dynamin, which contains the sequences needed for binding to both the clathrin-coated and non-clathrin-containing domains, nevertheless colocalized with clathrin to a very high degree (Fig. 1).

Our findings indicate that dynamin can bind to both clathrin-coated and non-clathrin-containing regions of the plasma membrane. Our observation that an SH3 domain ligand site is required for binding to coated pits suggests that dynamin and signal-transducing proteins may be coordinately recruited to the endocytic pathway. Further work will be needed to elucidate the identities of the specific ligands through which the interactions of dynamin with the plasma membrane are mediated.


FOOTNOTES

*
Parts of this work were previously reported in abstract form (Shpetner, H. S., Burgess, C. C., and Vallee, R. B.(1992) Mol. Biol. Cell3, 4 (abstr.); Shpetner, H. S., Vaughan, K. T., Burgess, C. C., and Vallee, R. B.(1993) Mol. Biol. Cell4, 171 (abstr.)). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address: Dept. of Cell Biology and Program in Molecular Medicine, University of Massachusetts Medical Center, 373 Plantation St., Worcester, MA 01605.

(^1)
The abbreviations used are: PH, pleckstrin homology; GTPS, guanosine 5`-3-O-(thio)triphosphate.


ACKNOWLEDGEMENTS

We thank Dr. Silvia Corvera for critically reading this manuscript.


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©1996 by The American Society for Biochemistry and Molecular Biology, Inc.