Structure-function analysis of SAP97, a modular scaffolding protein that drives dendrite growth

Abstract

Activation of AMPA receptors assembled with the GluA1 subunit can promote dendrite growth in a manner that depends on its direct binding partner, SAP97. SAP97 is a modular scaffolding protein that has at least seven recognizable protein-protein interaction domains. Several complementary approaches were employed to show that the dendrite branching promoting action of full length SAP97 depends on ligand(s) that bind to the PDZ3 domain. Ligand(s) to PDZ1, PDZ2 and I3 domains also contribute to dendrite growth. The ability of PDZ3 ligand(s) to promote dendrite growth depends on localization at the plasma membrane along with GluA1 and SAP97. These results suggest that the assembly of a multi-protein complex at or near synapses is vital for the translation of AMPA-R activity into dendrite growth.

Keywords: GluA1; PDZ domain; Spinal cord neuron.

Figure 1. Domain Structure of SAP97 constructs, expression levels, cellular distribution and effects on neuronal cell surface GluA1 abundance

Panel A. A cartoon depiction of recognizable protein-protein interaction domains of full length SAP97 (FL-SAP97) and the various truncation and point mutants employed in this study. Panel B. Western blots of various constructs expressed in HEK 293 cells. There are minor variations in the expression levels of individual constructs but no correlation between expression level and biological effects on dendrite growth. Panel C. Immunocytochemical localization of myc tagged PDZ3 shows that the protein is widely distributed throughout neuronal processes. GFP was used to delineate neuronal architecture and the myc-PDZ3 was imaged in the red channel. The merge image shows perfect co-localization. Calibration bar = 25 μm. Panel D. Quantification of cell surface GluA1 immunoreactivity in neurons expressing individual domains. The reported values are a ratio of GluA1 fluorescence to GFP (the co-transfection marker). In comparison with no domain controls, there were few differences in surface GluA1 abundance. Neurons expressing the SH3 domain had significantly more surface GluA1 in comparison with neurons expressing the PDZ2 or the I3 domain. It is noteworthy that expression of the SH3 domain was associated with more GluA1 expression but had no effect on dendrite growth (see table 5). Panel E. Expression levels of FL WT SAP97 and FL PDZ3 mutant SAP97 are very similar as are actin levels.

Figure 2. PDZ2 reduces the association of GluA1 with SAP97 although it (and the L27 and PDZ3 domains) does not associate directly with SAP97; the PDZ2 domain reduces dendrite growth

Panel A. After co-transfection of HEK 293 cells with GluA1 + SAP97 + either L27 or PDZ3 or PDZ2, cell lysates were prepared; half of the material was immunoprecipitated (IP'ed) with anti GluA1 and half was IP'ed with anti-SAP97. Aliquots from the initial lysate were reserved for input detection. In the upper panel, expression of PDZ2, but not PDZ3 or L27, reduces the abundance of the GluA1/SAP97 complex (IP GluA1, immunoblot (IB) SAP97). In the lower panel, SAP97 is found in the SAP97 immunoprecipitate (IB SAP97) but none of the domains are found in this material. Input blots show that the initial amount of all the proteins used in these experiments were virtually identical. Panel B. A single GFP labeled neuron, typical of cells studied in this work. All the labeled processes are dendrites with the exception of axon eminating from the cell body at the 4 o'clock orientation and denoted by the “^”. Calibration bar = 25 microns. Panel C. A camera lucida drawing of the neuron in Panel A. Panel D,1. Enlargement of the distal portion of dendrite outlined in a rectangle in Panel B. (the boxed region has been rotated ~ 20° clockwise). In addition to two branch points, several thin hairy appendages are visible. Calibration bar = 6 microns. Panel D,2. An outline of the dendrite in panel D,1 super imposed on the GFP labeled dendrite. Panel D,3. The outline in Panel D,2 has been moved onto a white background to better visualize the dendrite, two branch points, a dendritic branch of approximately 20 microns length and hairy appendages. Panel D,4. The camera lucida rendering of the dendrite outlined in Panels D,2 and D,3. Only two branch points are drawn; hairy appendages are not included in the final stick figure used in the quantitative dendrite analysis Panel E. Representative camera lucida drawings of neurons expressing GFP alone or with various other constructs as labeled. SAP97 overexpression promotes dendrite growth and branching and this is blocked by co-expression of PDZ2. Calibration bar = 25 μm. Panel F. Quantitative dendrite analysis using the “branches ≥ 4 micron criteria” indicates that PDZ2 blocks endogenous dendrite growth as well as the pro-dendrite growth action of SAP97. The “n” values in the first row indicate the number of individual neurons subjected to camera lucida drawing. Panel G. Quantitative dendrite analysis using the “branches ≥10 micron criteria” – the absolute values differ between the data in Panels F and G, the underlying observations match well.

Figure 3. RNA interference knockdown of WT but not resistant (“res”) versions of WT and PDZ3 mutant versions of full length SAP97; effects on dendrite growth

Panel Ai. Expression plasmids for WT or res full length SAP97 was co-transfected in HEK 293 cells with pSUPER construct that targeted SAP97 (pSUPER-SAP97) or a scrambled control (pSUPER scr). By western blot, pSUPER-SAP97 knocked down WT SAP97 but not res-SAP97. None of these constructs were toxic as evidenced by equivalent levels of actin. Panel Aii. Expression plasmids for WT or res full length PDZ3 mutant SAP97 was co-transfected in HEK 293 cells with pSUPER construct that targeted SAP97 (pSUPER-SAP97) or a scrambled control (pSUPER scr). By western blot, pSUPER-SAP97 knocked down WT PDZ3 mutant SAP97 but not res-PDZ3 mutant SAP97. None of these constructs were toxic as evidenced by equivalent levels of actin. Panel Aiii. HSV was engineered to express a control (“scr” for scrambled) or active (“SAP97 RNAi”) constructs and applied to spinal cord cultures. By western blot, the active RNAi construct reduced the abundance of SAP97 in comparison with the control. Neither construct was toxic as evidenced by equivalent levels of actin. Panel B. Quantitative dendrite analysis indicate that knockdown of SAP97 stunts dendrite growth and there is rescue to normal growth (that is, equivalent to expression of GFP alone) when knockdown is accompanied by expression of RNAi resistant WT SAP97. Panel C. Quantitative dendrite analysis indicate that rescue of dendrite growth abnormalities evoked by knockdown of SAP97 occurs with expression of RNAi resistant WT SAP97 but not PDZ3mut SAP97.

Figure 4. PDZ3 binding mutant SAP97 (PDZ3mut SAP97) does not stimulate dendrite branching; rescue by co-expression of palmitoylated PDZ3 (palPDZ3)

Panel A shows immunoblots for myc tagged palmitoylated and non-palmitoylated PDZ3 (palPDZ3 and PDZ3, respectively). Membranes were isolated from transfected cells by hypotonic lysis and centrifugation. Palmitoylated PDZ3 preferentially associates with membranes while non-palmitoylated PDZ3 is enriched in cytosolic fractions. Panel B shows representative camera lucida images of neurons expressing the noted constructs. Overexpression of PDZ3mut SAP97 has no effect on dendrite branching (in comparison with GFP only expressing neurons). Co-expression of PDZ3mut SAP97 with palPDZ3 rescues the dendrite growth promoting action of SAP97. Calibration bar = 25 μm. Panel C. There is a statistically significant increase in dendritic tree size and branching in neurons overexpressing PDZ3mut SAP97 + palPDZ3.

Figure 5. Simply localizing PDZ3 to the plasma membrane rescues the loss of SAP97 phenotype

Panel A. Western blots showing that an active pSUPER RNAi construct knocks down SAP97 but not GluA1. Panel B. Quantitative dendrite analysis indicates that endogenous SAP97 provides a limiting amount of substrate for palPDZ3 to operate upon.

Figure 6. GluA1 is required for the dendrite growth promoting effects of SAP97

Panel A. Western blots showing that an active pSUPER RNAi construct knocks down GluA1 but not SAP97 or GluA2. Panel B. Quantitative dendrite analysis inciates that knockdown of GluA1 reduces dendritic branching and overall tree size compared to controls and prevents dendrite growth evoked by overexpression of SAP97. Panel C. Quantitative dendrite analysis indicates that GluA1 is required for SAP97-dependent dendrite growth both because it localizes SAP97 to synapses and because it provides agency. The nature of this second activity is likely to be linked to calcium permeability (Jeong et al., 2006).