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    • We have shown that an extensive region in SERBP

    2023-09-11

    We have shown that an extensive region in SERBP1 (amino acids 354 to 474, using co-ordinates based on the full-length isoform, Figs. 1a and 2) is necessary for it to interact with RACK1; since this region has substantial homology to the corresponding region of HABP1 (Fig. 3) it is a reasonable hypothesis that this region of HABP4 is also essential for it to interact with RACK1. More generally, it appears that many of RACK1's partners employ a large region(s) of their respective proteins in their interaction with RACK1; for example, we and our collaborators have identified 2 extensive regions in PDE4D5 that are required for it to interact with RACK1 [10], [11], [12], [13], [14], [15], [16], [95]. The large size of the interacting regions of PDE4D5 and RACK1 is consistent with the high avidity of their interaction; in this regard, we and our collaborators have shown previously that the interaction between RACK1 and PDE4D5 has an affinity in the low nanomolar range [10]. Although there is no obvious amino flavin adenine dinucleotide receptor sequence homology between the RACK1-interaction regions of PDE4D5 and that of SERBP1 (and, by extension, that of HABP4), it is possible that similarities in the overall protein conformation of these regions might be apparent upon appropriate structural study. Given the large region(s) on RACK1's partners that seem to be essential for many of them to interact with RACK1, a reasonable hypothesis would be that a corresponding large region on RACK1 is also essential for the interaction. We have shown previously that a large surface (WD-repeats 5 through 7) on RACK1 is essential for it to interact with PDE4D5 and have identified a number of amino acids on RACK1 that, when individually mutated, markedly attenuate their interaction [12]. In the present study, we have shown that many of these RACK1 mutants also attenuate its interaction with SERBP1 (Fig. 4). These observations suggest, but do not prove, that a large region of RACK1 interacts with SERBP1 and that this region overlaps substantially with the region of RACK1 that is essential for it to interact with PDE4D5. Potential alternative explanations for our data could be that some of the mutations alter the conformation of RACK1 generally (rather than affecting the interaction face itself), or destabilize the protein in cells. Finally, we cannot exclude the possibility that some of our observations reflect artifacts of the yeast 2-hybrid system; however, our S. cerevisiae-based system has, in a very large number of cases, been shown to be a reasonably physiologic system for these studies. One important issue in interpreting data on the interaction of RACK1 and its partners is the stoichiometry of the complex. In our 2-hybrid studies, RACK1 and its partners each interact as monomers. Some studies have suggested that RACK1 is dimeric in cells [96], [97], [98], [99], while other data [100] are more consistent with its existing in solution primarily as a monomer. It is clearly monomeric in the ribosome [101]. If RACK1 exists primarily as a monomer in cells, then our mutagenesis data are consistent with the surface on WD repeats 5 through 7 directly interacting with PDE4D5 and SERBP1; however, if RACK1 exists as a dimer, then the mutations could disrupt RACK1 dimerization and not affect the actual interaction site(s) with its partners. Additional structural or interaction data will be essential to distinguish these possibilities. Finally, we cannot exclude the possibility that many of RACK1's partners could also dimerize or multimerize; in this regard, we, our collaborators, and a number of other groups have clearly shown that PDE4D5 can dimerize [83], [84], [102], [103], [104], [105], although the regions of PDE4D5 that are essential for dimerization are quite separate from those necessary for it to interact with RACK1 (see ref. [17] for a more complete discussion). Our 2-hybrid screen data also provide some additional insight into the interaction of RACK1 with the small (40S) ribosomal subunit. A large number of biochemical studies have shown previously that RACK1 interacts directly with the 40S subunit. A structural study employing X-ray diffraction has shown that RACK1 interacts directly with rpS16e, rpS17e, and rpS3e [new names: uS9, eS17 and uS3, respectively, ref. [59]] and the 18S rRNA of the human 40S subunit [101]. In our 2-hybrid screen, we isolated fusions encoding the rpS13e and rpS17e proteins (new names uS15 and eS17, respectively; ref. [59]). Our data confirm the importance of rpS17e in the interaction of RACK1 with the 40S subunit; however, the significance of our rpS13e isolate is unclear, as this protein is located some distance away from RACK1 [101]. The functional significance of the interaction of RACK1 with the ribosome, especially in complex eukaryotes, remains uncertain [3].