An Exon Splice Enhancer Primes IGF2:IGF2R Binding Site Structure and Function Evolution

Abstract

Placental development and imprinting co-evolved with parental conflict over resource distribution to mammalian offspring. The imprinted genes, IGF2 and IGF2R, code for the growth promoter insulin-like growth factor 2 and its binding inhibitor, mannose 6-phosphate/IGF2 receptor, respectively. M6P/IGF2R of birds and fish do not recognize IGF2. In monotremes that lack imprinting, IGF2 specifically bound M6P/IGF2R via a hydrophobic CD loop. We show that the DNA coding the CD loop in monotremes functions as an exon splice enhancer (ESE) and that structural evolution of binding site loops (AB, HI, FG) improved therian IGF2 affinity. We propose that evolution of this ESE led to the fortuitous acquisition of M6P/IGF2R IGF2 binding that drew IGF2R into parental conflict prior to imprinting, that may have accelerated subsequent affinity maturation. † The sequence of molecular evolutionary events that established placental viviparity, genomic imprinting and parental conflict in mammals remain poorly understood (1) . Genomic imprinting occurs when expression of one allele of a diploid gene is silenced depending on the parent-of-origin, e.g. either from the father or the mother. Parental conflict over the distribution of resources to offspring has been supported by the observation of reciprocal imprinting of genes coding for the growth promoter Insulin-like growth factor 2 (IGF2), and the cation-independent mannose 6-phosphate/ IGF2 receptor (M6P/IGF2R or IGF2R) (2) . IGF2 and IGF2R are two of the approximately 80 genes imprinted in mammals, and two of the five genes (with INS, MEST/PEG1 and PEG10) imprinted in marsupials. So far, no evidence supports the existence of imprinting in monotremes despite the presence of a chorio-vitelline placenta (3, 4). On the basis of functional data, IGF2R transports M6P modified acid hydrolases to the pre-lysosomes (5). Of the 15 extra-cellular domains of IGF2R, domain 11 binds IGF2 in therians, and internalizes the ligand for degradation, whereas M6P bind to domains 3, 5 and 9 (5). Igf2 rescues placental dependent embryonic lethality associated with laboratory created murine parthenogenesis, implicating IGF2 supply as a regulator of placental development (6). Disruption of the maternal Igf2r allele results in Igf2 dependent overgrowth and fatality, supporting that IGF2R antagonizes the function of IGF2 (7,8). The structure of the unbound human domain 11 shows that the IGF2 binding site composed of defined loops (AB, CD, FG and HI, Fig. 1A and Fig. S1) but how this domain 11 evolved to bind IGF2, and the relationship to imprinting co-evolution, remain unknown (9-12).We established a high resolution structure of the human IGF2R:IGF2 complex and then compared this to other phylogenetically informative vertebrates. We adopted an NMR approach as the side chain amino acid interactions across the binding interface were not resolved in our 4.1Å resolution co-crystal structures (9). Wild-type human domain 11 and IGF2 failed to form a stable association in initial NMR studies. However, we ...

The Late Endosomal Transporter CD222 Directs the Spatial Distribution and Activity of Lck

“…The latter could be accomplished via regulating the bioavailability of the mitogenic insulin-like growth factor 2 that is both postnatally expressed in thymic epithelial cells (62) and upregulated in diseases (28), namely via internalization and subsequent degradation in lysosomes (12,63). 4) Furthermore, CD222's surface upregulation might play a role in T cell differentiation from the double negative to the double positive stage as CD222 Ab was reported to block T cell ontogeny (64).…”

Rapid molecular evolution across amniotes of the IIS/TOR network

“…We found that IGF2R has been shaped by putatively strong positive selection within reptiles and positively selected sites clustered on the IGF2R protein surface in domain 11, which is intimately involved in binding IGF1 and IGF2. Several of the positively selected sites on the protein surface of IGF2R in reptiles are essential for binding IGF2 based on mutagenesis studies and the crystal structure of the IGF2R-IGF2 complex (e.g., Y1542) (43)(44)(45) (Fig. 2C and Table S4).…”

“…Labeled sites (IGF1 S37 and N251; human numbering) are known to affect IGF hormone and receptor binding (Table S4). (C) Domain 11 of reptile and mammal IGF2R with IGF2 oriented toward the binding pocket to demonstrate the clustering of positively selected sites on the reptile IGF2R binding surfaces (43,44). The magenta sites on reptile IGF2 and IGF2R were identified as coevolving amino acids using CAPS (46).…”

Functional evolution of IGF2:IGF2R domain 11 binding generates novel structural interactions and a specific IGF2 antagonist

“…One such evolutionary biochemical example is the initial acquisition and subsequent gain of affinity between the insulin-like growth factor 2 (IGF2) ligand and a single domain of a nonsignaling mannose 6-phosphate (M6P)/IGF2 receptor (IGF2R) (domain 11). The structural and functional basis of this evolutionary path, which has occurred over 150 million years of mammalian evolution, has been reported previously (2). The questions that we address in the present work are whether the IGF2:domain 11 interaction has reached an optimal state in the context of IGF2 activation of signaling receptors and in the ligand clearance function of M6P/IGF2R, and how far can we extend the binding interaction in terms of structural, biophysical, and functional properties.…”

“…Domain 13 indirectly interacts with the AB loop of domain 11, breaking this interaction in the multiple-domain receptor, and contributes to the stability of the complex by decreasing the "off-rate" (k off ) of the IGF2 interaction (21). Mutations in the AB loop replace the effect of domain 13, increase the affinity for IGF2 (2,22), and facilitate subsequent determination of a solution structure of the 24.2-kDa complex of IGF2 with domain 11 AB3 that incorporated three mutations of the AB loop (also known as AB3 or E1544K, K1545S, and L1547V; K D = 15 nM) (2). The structural basis of the IGF2:domain 11 AB3 interaction occurs with a relatively fixed conformation of the CD loop on complex formation.…”