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Crucial Role of the SH2B1 PH Domain for the Control of Energy Balance

Disruption of the adaptor protein SH2B1 (SH2-B, PSM) is associated with severe obesity, insulin resistance, and neurobehavioral abnormalities in mice and humans. Here, we identify 15 SH2B1 variants in severely obese children. Four obesity-associated human SH2B1 variants lie in the Pleckstrin homology (PH) domain, suggesting that the PH domain is essential for SH2B1’s function. We generated a mouse model of a human variant in this domain (P322S). P322S/P322S mice exhibited substantial prenatal lethality. Examination of the P322S/+ metabolic phenotype revealed late-onset glucose intolerance. To circumvent P322S/P322S lethality, mice containing a two-amino acid deletion within the SH2B1 PH domain (ΔP317, R318 [ΔPR]) were studied. Mice homozygous for ΔPR were born at the expected Mendelian ratio and exhibited obesity plus insulin resistance and glucose intolerance beyond that attributable to their increased adiposity. These studies demonstrate that the PH domain plays a crucial role in how SH2B1 controls energy balance and glucose homeostasis.

Hyperphagia, severe obesity, insulin resistance, and neurobehavioral abnormalities have been reported in individuals with rare coding variants in the gene encoding SH2B1 (SH2-B, PSM) (1,2). Consistently, mice null for Sh2b1 exhibit obesity, impaired glucose homeostasis, and often, aggressive behavior (3)(4)(5). Transgenic expression of the b-isoform of SH2B1 (SH2B1b) in the brain largely corrects the obesity and glucose intolerance of otherwise Sh2b1-null mice (6), suggesting the importance of brain SH2B1 for the control of energy balance and glucose homeostasis.
At the cellular level, SH2B1 is an intracellular adaptor protein that is recruited to phosphorylated tyrosine residues on specific membrane receptor tyrosine kinases (e.g., receptors for brain-derived neurotrophic factor [BDNF], nerve growth factor [NGF], insulin) and cytokine receptor/ Janus kinase (JAK) complexes (e.g., leptin receptor/JAK2) and enhances the function of these receptors (7)(8)(9)(10)(11)(12)(13). The exact mechanism(s) by which it does so is unclear, although a variety of mechanisms have been proposed. These include enhanced dimerization causing increased activation of the kinase itself (14), stabilization of the active state of the kinase (15), decreased dephosphorylation or increased complex formation of insulin receptor substrate (IRS) proteins bound to receptors or receptor/JAK2 (16,17), regulation of the actin cytoskeleton (18), and activation of specific pathways, including extracellular signal-regulated kinases (ERKs), Akt, and/or phospholipase Cg (10,19). Some of these receptors, including the leptin, BDNF, and insulin receptors, play important roles in the central control of energy expenditure and/or glucose homeostasis (20). SH2B1b has been shown to enhance BDNF-and NGF-stimulated neurite outgrowth in PC12 cells (13,21).
The four isoforms of SH2B1 (a, b, g, d), which differ only in their COOH termini, share 631 NH 2 -terminal amino acids. These amino acids possess a dimerization domain, Pleckstrin homology (PH) domain, src-homology 2 (SH2) domain, nuclear localization sequence (NLS), and nuclear export sequence (NES) (22)(23)(24) (Fig. 1A). The SH2 domain enables SH2B1 recruitment to specific phosphorylated tyrosine residues in activated tyrosine kinases (25). The NLS and NES are essential for SH2B1 to shuttle among the nucleus, the cytosol, and the plasma membrane (22,23). The NLS combined with the dimerization domain enables SH2B1 to associate with the plasma membrane (26). However, the function and importance of the SH2B1 PH domain remains largely unknown. Four human obesity-associated variants lie in the SH2B1 PH domain (Fig. 1A), suggesting the importance of the PH domain in SH2B1 function. The PH domains of some proteins bind inositol phospholipids to mediate membrane localization (27,28). However, 90-95% of all human PH domains do not bind strongly to phosphoinositides and presumably mediate other functions (29). Indeed, the PH domain of SH2B1 neither localizes to the plasma membrane nor is required to localize SH2B1b to the plasma membrane (23,30). Here, we tested the importance of the PH domain of SH2B1 in vivo by generating and studying mice containing human obesity-associated (P322S) or engineered (inframe deletion of P317 and R318 [DPR]) mutations in the SH2B1 PH domain. Our results demonstrate that the SH2B1 PH domain plays multiple crucial roles in vivo, including for the control of energy balance and glucose homeostasis, and in in vitro studies, changes the subcellular distribution of SH2B1b and enhances NGF-stimulated neurite outgrowth in PC12 cells.

Human Studies
The Genetics of Obesity Study (GOOS) is a cohort of .7,000 individuals with severe obesity with age of onset of ,10 years (31,32). Severe obesity is defined as a BMI (kg/m 2 ) SD score .3 (U.K. reference population). Whole-exome sequencing and targeted resequencing were performed as in Hendricks et al. (33). All variants were confirmed by Sanger sequencing (1). HOMA of insulin resistance (HOMA-IR) was calculated using the equation HOMA-IR score 5 [(fasting insulin in mU/mL) 3 (fasting glucose in mg/dL)] / 405, which estimates steady-state b-cell function and insulin sensitivity (34,35). All human studies were approved by the Cambridge local research ethics committee. Each subject (or parent for those ,16 years of age) provided written informed consent; minors provided oral consent. Mouse Models, Genotyping, and Gene Expression CRISPR/Cas9 genome editing was used to insert the P322S mutation into mice. The reverse complement of the genomic Sh2b1 sequence in C57BL/6J mice (accession number NC_000073, GRC m38) was used to design the reagents for CRISPR. The guides were designed using the website described in Ran et al. (36). The mutations in the donor are summarized in Fig. 1C

Mouse Body Weight and Food Intake
Mice were individually housed, and body weight and food consumption were assessed weekly.

Mouse Glucose Tolerance Tests, Insulin Tolerance Tests, and Hormone Levels
Mice were fasted 0900-1300 h for glucose tolerance tests (GTTs) or 0800-1400 h for insulin tolerance tests (ITTs). Glucose or human insulin was injected intraperitoneally, and blood was collected from the tail vein. Blood glucose levels were assessed using a Bayer Contour glucometer. For plasma insulin levels, mice were fasted 0800-1400 h, and tail blood was assayed using an Ultra Sensitive Mouse Insulin ELISA kit (#90080; Crystal Chem). Tail blood (0900-1000 h) (Figs. 2F and 4G) or trunk blood after sacrifice (1000-1300 h) (Fig. 6B) from fed mice was tested for leptin using a Mouse Leptin ELISA kit (#90030; Crystal Chem).

Mouse Body Composition, Metabolic Assessment, and Tissue Collection
Body composition was measured at room temperature in the morning or evening (Fig. 6A) using a Minispec LF90 II Bruker Optics nuclear magnetic resonance (NMR) analyzer (University of Michigan Animal Phenotyping Core). To assess metabolic state, mice were single-housed for 3 days and then tested for 72 h using a Comprehensive Lab Animal Monitoring System (Columbus Instruments). O 2 consumption (VO 2 ), CO 2 production (VCO 2 ), X activity, and Z activity were collected in 20-min bins. The final 24 h of recordings are presented. Mice were sacrificed . Amino acid residues for newly characterized human obesity-associated SH2B1 variants and the previously characterized variant P322S are shown. DD, dimerization domain; P, proline-rich region. B: SH2B1 mutations impair the ability of SH2B1 to enhance neurite outgrowth. PC12 cells transiently coexpressing GFP and either empty pcDNA3.1(1) vector (2), human SH2B1b WT, or SH2B1b mutant were treated with 20 ng/mL rat NGF for 3 days, after which neurite outgrowth was assessed. R555E lacking an intact SH2 domain and the human mutation P322S reported previously were included as positive controls. GFP-positive cells were scored for the presence of neurites two times the length of the cell body ($400 cells/condition/experiment). The percentage of cells with neurites was determined by dividing the number of GFP-positive cells with neurites by the total number of GFP-positive cells. Data are mean 6 SEM (n 5 5). Each construct was compared with WT using a two-tailed Student t test. *P , 0.05, **P , 0.01, ***P , 0.001, ****P , 0.0001. C: CRISPR/Cas9 schematic for Sh2b1 P322S gene editing. RNA guide sequence, PAM sequence, and cut site for Cas9 are shown for guide 2. The region of the 180-nucleotide oligo donor template used to direct homology-directed repair in the vicinity of the DPR deletion and P322 is shown. Mutations in donor template that introduce the C.T mutation to code for P322S and silent mutations to create a diagnostic XbaI site and disrupt guide RNA binding following repair are highlighted in red. aa seq., amino acid sequence. D: Proteins in brain tissue lysates from Sh2b1 WT, P322S/P322S, and KO male mice were immunoblotted with aSH2B1 or ab-tubulin. Migration of the 100-kDa protein standard and the four isoforms of SH2B1 are shown. IB, immunoblot. E: P322S/P322S mice from intercrosses of heterozygous mice were born at approximately one-half of the expected Mendelian ratio. P , 0.05, x 2 test. n 5 179 mice.
(1000-1300 h) using decapitation under isoflurane. Trunk blood was collected and the serum stored at 280°C. Tissues were collected, weighed, cryopreserved in liquid nitrogen, and stored at 280°C.

Live Cell Imaging
The indicated construct was transiently transfected into 293T cells or PC12 cells. Cells were treated and live cell images captured by confocal microscopy using an Olympus FV500 laser scanning microscope and FluoView version 5.0 software, as in Joe et al. (19).

Structural Modeling and ClustalW Analysis
A structural model for human SH2B1 was created by overlaying the PH domain sequence of human SH2B1 onto the mouse NMR structure of APS (Protein Data Bank ID 1V5M) using the PyMOL Molecular Graphics System version 2.2.3. ClustalW alignments were performed using LaserGene version 14.0.0 (DNASTAR, Madison, WI). Functional homology was defined as residues that match the consensus within 1 distance unit using the PAM250 mutation probability matrix.

Statistics
All nonhuman analyses were carried out using GraphPad Prism software. Body weight, GTTs, and ITTs were analyzed by two-way ANOVA followed by Fisher least significant difference posttest. Food intake was analyzed by linear regression. Significance of the deviation of birth rate from the expected Mendelian ratio was assessed using x 2 test. For other physiological parameters, experimental animals were compared with their wild-type (WT) littermates by two-tailed Student t test. Neurite outgrowth was analyzed by a two-tailed Student t test. For all comparisons, P , 0.05 was considered significant.

Data and Resource Availability
Any raw data sets generated during the current study are available from the corresponding author on reasonable request, with all reagent and analytical details included in the published article (and its Supplementary Data). The mouse models generated and analyzed during the current study are available from the corresponding author upon reasonable request.

Identification and Characterization of 15 Rare Human Variants in SH2B1
Using exome sequencing, targeted resequencing, and Sanger sequencing of 3,000 individuals exhibiting severe obesity before the age of 10 years (33), we identified 15 rare variants in SH2B1 in 16 unrelated individuals (Table 1 and Fig. 1A). Eleven variants are newly identified, while four (R227C, R270W, E299G, V209I) have been previously reported in other obese individuals but not well characterized (4,38,39). Fourteen of these variants are in the first 631 amino acids shared by all isoforms of SH2B1. The mean 6 SD BMI SD score of variant carriers was 4.0 6 0.6. The 15th variant causes the G638R mutation in the COOH-terminal tail unique to the b-isoform of SH2B1. A number of the SH2B1 variant carriers had HOMA-IR (34,35) scores of .1.9, indicating insulin resistance and increased risk of type 2 diabetes (40). Some of the HOMA-IR scores, including those for people carrying three variants in or near the PH domain (G238C, R270Q, and M388V), were particularly high. A spectrum of neurobehavioral abnormalities, including learning difficulties, dyspraxia, hyperactivity/inattention, aggression/emotional lability, anxiety, and autistic traits, were detected in all the individuals for whom behavioral information was available (Table 1). In the neurite outgrowth assay, 7 of these 15 rare variants impaired the ability of SH2B1b to stimulate NGF-induced neurite outgrowth (Fig. 1B), suggesting that many of the variants negatively affect the neuronal function of SH2B1. Because the variants are found in multiple domains in SH2B1 and throughout the SH2B1 sequence, it is not surprising that individuals with different SH2B1 variants have different phenotypes. These newly characterized variants add support to SH2B1 being an important regulator of human body weight, insulin sensitivity, and behavior. Interestingly, four of the human obesityassociated SH2B1 variants lie in the PH domain of SH2B1, suggesting the importance of the PH domain in the ability of SH2B1 to regulate energy balance, glucose metabolism, and behavior.   (51); 0, variant was detected in gnomAD exomes with MAF 5 0 in the NFE sample; *, variant not present in NFE samples but present in samples from other ethnic groups; †, variant not found in any gnomAD exomes; ‡, homozygous variant in a proband from a consanguineous family. F, female, M, male. BMI is weight in kg/height in m 2 with ageand sex-adjusted BMI SD score shown in parentheses. When fasting plasma glucose and insulin were available, HOMA-IR was calculated. Scores in young obese children are difficult to interpret given the paucity of control data. However, in adults, HOMA-IR .1.9 indicates insulin resistance. Behavioral data were gathered from medical history. 1 indicates presence of a phenotype. 2 indicates absence of a phenotype. S indicates speech and language delay. Blank cells indicate insufficient information to record a phenotype.

Developmental Lethality in Mice Homozygous for the SH2B1 P322S Mutation
To gain insight into the role of the PH domain of SH2B1 in energy balance and glucose metabolism, we studied the effect of the P322S human obesity-associated SH2B1 PH domain variant in mice. We chose the P322S variant because of its strong association with obesity in the proband family (1), the conservation of P322 across mammals and with the SH2B1 family member SH2B2/APS, its predicted disruptive effect on PH domain function by Provean (RRID:SCR_002182) and PolyPhen (RRID:SCR_013189) analysis, and P322S-dependent deficiencies in SH2B1 function observed in cultured cells (1). We used CRISPR/Cas9-based genome editing to introduce the P322S variant into Sh2b1 in C57BL/6J mice (Fig. 1C). DNA sequencing confirmed germline transmission of the P322S edit ( Supplementary Fig. 1A). The P322S mutation neither affects the mRNA levels for any of the Sh2b1 isoforms in the examined tissues (brain, liver, and heart) (Supplementary Fig. 1B and C) nor alters SH2B1 protein levels or isoform selection in brain tissue ( Fig. 1D and Supplementary Fig. 1D). However, we found that homozygous (P322S/P322S) mice are born at much less than the expected Mendelian ratio (Fig. 1E), suggesting that the P322S mutation disrupts SH2B1 PH domain function in a manner that interferes with embryo implantation and/or development. Consistent with this, preliminary data from timed pregnancies reveal that at embryonic day 17, the homozygous embryos (P322S/ P322S) are also present at less than the expected Mendelian ratio.

Mice Heterozygous for P322S Exhibit Altered Glucose Tolerance, but Not Altered Energy Balance
In addition to the difficulty of producing sufficient P322S/ P322S mice for study, the high rate of embryonic lethality in P322S/P322S mice suggested that the surviving P322S/ P322S mice might have underlying poor health, which could interfere with the analysis of their metabolic phenotype. For these reasons, and because human obesity is linked with heterozygosity for P322S (1), we studied energy balance and glucose homeostasis in heterozygous (P322S/1) male (Fig. 2) and female ( Supplementary Fig. 2) mice. We found no difference in food intake, body weight, or adiposity between WT and P322S/1 mice fed standard chow (9% fat) or an HFD (60% fat). However, in contrast to their WT littermates, 28-week-old HFD-fed P322S/1 male and female mice displayed glucose intolerance in an intraperitoneal GTT. Neither insulin concentrations nor the response to an ITT were altered in the P322S/1 animals compared with littermate controls, however. These findings suggest that the PH domain of SH2B1 is important for SH2B1 function, including for the control of glucose homeostasis, but that the resultant metabolic phenotype is less penetrant in the heterozygous state in mice than it is in humans. We thus sought to study mice homozygous for mutations in the SH2B1 PH domain.

Deletion of P317 and R318 in the PH Domain Alters the Subcellular Localization of SH2B1
Because of the early lethality of P322S/P322S mice, we examined the function of another SH2B1 mutation containing a two-amino acid deletion (DPR) within the PH domain of SH2B1 ( Fig. 1C and Supplementary Fig. 1E). This mutation arose as a separate line during the generation of the P322S mice.
When transiently expressed as green fluorescent protein (GFP) fusion proteins in PC12 cells, SH2B1b and SH2B1b DPR demonstrated similar expression levels (Fig.  3A), suggesting that DPR does not destabilize the protein.
However, while GFP-SH2B1b localizes primarily to the plasma membrane and cytoplasm in 293T and PC12 cells (as previously shown [22,26]), SH2B1b DPR localizes primarily to the nucleus ( Fig. 3B and C). The nuclear localization of SH2B1b DPR suggests that the DPR mutation alters SH2B1 nuclear cycling to favor retention in the nucleus. We predicted that this altered localization would change the cellular function of SH2B1b DPR. Indeed, SH2B1b-dependent NGFstimulated neurite outgrowth in PC12 cells was decreased in cells expressing SH2B1b DPR (Fig. 3D). Thus, disruption of the PH domain by the DPR mutation alters the subcellular distribution of SH2B1b and impairs the ability of SH2B1b to enhance neurotrophic factor-induced neurite outgrowth.

Obesity, Hyperphagia, and Disrupted Glucose Homeostasis in Mice Homozygous for the SH2B1 DPR Mutation
We examined the phenotype of the mice containing the DPR mutation with the hope that this mutation might produce a less dramatic reproductive phenotype than that observed with P322S in the homozygous state, allowing us to examine the effects of the DPR mutation on energy balance and glucose homeostasis in homozygous mice. As with the P322S mutation, DPR did not affect the mRNA levels for any of the Sh2b1 isoforms in the tissues tested (brain or heart) (Fig. 4A). At the protein level, the DPR mutation did not alter the relative levels of the different isoforms in brain tissue, although levels of SH2B1 protein were somewhat reduced (Fig. 4B). Importantly, in contrast to P322S/P322S mice, DPR/DPR homozygous mice were born and survived at the expected Mendelian frequency (Supplementary Fig. 1F), permitting us to examine the effect of this SH2B1 PH domain mutation in the homozygous state.
DPR/DPR mice fed standard chow exhibit significantly increased body weight compared with their WT littermates ( Fig. 4C and D). By 20 weeks of age (Fig. 4C), DPR/DPR male mice were 15 g (.40%) heavier than their WT littermates, while female DPR/DPR mice were ;9 g (;35%) heavier than their WT littermates. It should be noted that we do not believe that the reduced levels of SH2B1 protein in the DPR/DPR mice can account for the increased obesity detected in DPR/DPR mice because heterozygote Sh2b1 2/1 mice are not obese (5). Body length was not significantly different in preliminary studies (Supplementary Fig. 1G and H). Overall adiposity (Fig. 4E and F) as well as circulating leptin concentrations (Fig. 4G) were increased in DPR/DPR homozygotes but not lean body mass (Fig. 4H). The heterozygous (DPR/1) male and female mice showed no significant increase in adiposity (Fig. 4F). However, DPR/1 males had a slight increase in circulating leptin levels (Fig. 4G), suggesting that in males, even a single copy of the DPR mutation may be sufficient to produce a minor effect on energy balance.
Increased food intake (assessed at 18-20 weeks) is observed in DPR/DPR male and female mice compared with their WT and DPR/1 littermates (Fig. 5A), while VO 2 (at 11-12 weeks of age) (Fig. 5B), respiratory exchange ratio (data not shown), and locomotor activity (data not shown) were not altered. On the basis of these data and the previous finding that Sh2b1 KO mice are obese primarily as a consequence of increased food intake (5), we believe it most likely that the DPR mutation caused obesity in the mice primarily as a consequence of increasing food intake rather than decreasing energy expenditure.
diabetes.diabetesjournals.org Flores and Associates glucose tolerance (Fig. 5D), although other parameters of glucose homeostasis were not different from WT littermates.
Because the disruption of glucose homeostasis in the aged DPR/DPR mice presumably resulted (at least in part) from their increased adiposity, we examined glucose Figure 6-DPR female mice exhibit reduced glucose tolerance before the onset of obesity. A: Body fat mass was determined at weeks 11-12. Percent fat mass was determined by dividing the mass by body weight (males: n 5 13 WT, 10 DPR/1, 11 DPR/DPR; females: n 5 11 WT, 15 DPR/1, 13 DPR/DPR). B: At week 7, serum from WT, DPR/1, and DPR/DPR male mice was assayed for leptin (males: n 5 8 WT and DPR/DPR, 7 DPR/1; females: n 5 8 WT, 5 DPR/1, 12 DPR/DPR). C: Eight-week-old mice were fasted for 6 h, and insulin levels were determined (males: n 5 11 WT, 9 DPR/1, 10 DPR/DPR; females: n 5 9 WT, 11 DPR/1, 13 DPR/DPR). D: At week 8, mice were fasted for 4 h, and blood glucose was measured (males: n 5 13 WT, 10 DPR/1, 11 DPR/DPR; females: n 5 10 WT, 13 DPR/1, 12 DPR/DPR). E: In a separate study, GTT was assessed at 8 weeks as in Fig. 2D and blood glucose monitored at the times indicated (males: n 5 13 WT, 10 DPR/1, 11 DPR/DPR; females: n 5 10 WT, 13 DPR/1, 12 DPR/DPR). F: ITT was assessed at 9 weeks as in Fig. 2E and blood glucose monitored at the times indicated (males: n 5 11 WT, 9 DPR/1, 10 DPR/DPR; females: n 5 9 WT, 11 DPR/1, 13 DPR/DPR). Same cohort of mice as Fig. 5B. For all comparisons, data are mean 6 SEM. *P , 0.05, **P , 0.01, ***P , 0.001, ****P , 0.0001 compared with WT littermates. n.s., not significant. homeostasis in young preobese mice to define any adiposity-independent effects of SH2B1 DPR on glucose homeostasis. We examined the adiposity of younger DPR/DPR mice to determine an age at which we might examine glucose homeostasis without it being confounded by increased adiposity. At 11-12 weeks of age, adiposity was already increased in male DPR/DPR mice but not detectably increased in female DPR/DPR mice (Fig. 6A). By 7 weeks, leptin levels were increased in male, but not female, DPR/DPR mice (Fig. 6B). We thus examined glucose homeostasis in DPR/DPR mice at 8 weeks of age, revealing hyperinsulinemia and glucose intolerance (with unchanged insulin tolerance) in both male and female DPR/DPR mice (Fig. 6C-F). The hyperinsulinemia and glucose intolerance in the presence of unchanged leptin and adiposity in young preobese females suggest that the DPR mutation interferes with glucose homeostasis independently of adiposity. Taken together, our results suggest that in obese DPR mice, the DPR mutation likely interferes with glucose homeostasis both independently of adiposity and secondary to the effects of adiposity on energy balance.

DISCUSSION
The identification of four human obesity-associated variants in the PH domain-encoding region of SH2B1, the fact that the three individuals with PH domain variants whose behavior has been documented all displayed behavioral abnormalities (1,2, and the present study), and the fact that the three individuals with variants in or near the PH domain had HOMA-IR scores suggesting severe insulin resistance and risk of type 2 diabetes, highlight the importance of the PH domain in SH2B1 function. The lethality of the human obesity-associated P322S mutation in the PH domain of SH2B1 in homozygous P322S/P322S mice demonstrates the importance of this mutation for SH2B1 function in vivo. Similarly, the obesity and diabetes observed in DPR/DPR mice highlight the importance of the SH2B1 PH domain for SH2B1-mediated metabolic control. The adiposityindependent glucose intolerance of young DPR/DPR female mice before the onset of obesity as well as of P322S/1 and DPR/1 mice also reveals the importance of SH2B1 and its PH domain for the control of glucose homeostasis independent of body weight, as previously suggested from the phenotype of humans bearing mutations in SH2B1 (1,2).
On the basis of the increased food intake of DPR/DPR mice and the findings in the Sh2b1 KO mice (5), we believe it likely that the increased body weight of the DPR/DPR mice is due to impaired function of SH2B1 in the hypothalamus. However, SH2B1 is also expressed in the periphery. The increased insulin concentrations with glucose intolerance in young, nonobese female DPR/DPR mice suggest alterations in tissues that control glucose uptake. However, the presence of glucose intolerance despite the increased insulin levels is consistent with the islets of DPR/DPR mice having an impaired ability to fully compensate for those alterations (41,42).
While humans heterozygous for P322S exhibit severe obesity, P322S/1 mice display mild glucose intolerance only in aged, HFD-fed animals. Because the region surrounding P322 is conserved between mice and humans Figure 7-ClustalW analysis and modeling of the three-dimensional structure of the PH domain of SH2B1. ClustalW of SH2B1, SH2B2/APS, and SH2B3/Lnk in the region included in the NMR structure of SH2B2/APS. Homologous residues are highlighted in black, and functionally homologous residues are cyan. The PH domain is indicated by the blue line below the sequences. P317, R318 in SH2B1 and the residues in SH2B1 for which variants are associated with obesity are indicated by magenta ovals. The variants in Lnk associated with myeloproliferative neoplasms are indicated by orange ovals. The variants are noted above the ClustalW. Residues within 8 Å of P317 in SH2B1 (P240 in SH2B3/ Lnk) are denoted by taupe ovals. Residues within 8 Å of R318 in SH2B1 (K241 in SH2B3/Lnk) in the 3-dimensional structure (Video 1) are denoted by purple ovals. Residues within 8 Å of P322 in SH2B1 (P245 in SH2B3/Lnk) are denoted by green ovals. hum, human; mus, mouse.
( Fig. 7), it is unlikely that the more modest phenotype of P322S/1 mice compared with humans reflects species differences that result in structural changes in the SH2B1 PH domain, per se, but rather that PH domain binding partners may have different tolerances for P322S in mice and humans and/or that human physiology adapts more poorly to the resultant alterations in SH2B1 function. Consistent with the importance of the PH domain for the function of SH2B family members, at least nine point mutations (E208Q/E, A215V, G220V/R, A223V, G229S, D234N, F287S) have been identified in the PH domain of the SH2B1 ortholog SH2B3/Lnk in patients with myeloproliferative neoplasms (43-48) (Fig. 7).
To gain insight into how the SH2B1 P322S mutation or deletion of residues P317 and R318 in SH2B1 might regulate the function of the PH domain in SH2B1, we performed ClustalW analysis of SH2B family members and analyzed a model of SH2B1 that was based on the NMR structure of the PH domain of the SH2B1 family member SH2B2/APS (49). ClustalW analysis of the PH domains of the SH2B family members reveals that the PH domains are highly conserved (Fig. 7). In the model, residues P317, R318, and P322S in SH2B1 are on an exterior surface of the PH domain (Video 1). This surface is presumably a binding interface that interacts with either another region in SH2B1 or another protein.
Another human obesity-associated variant in SH2B1 (E299G) as well as five of the human myeloproliferative neoplasm-associated variants in SH2B3/Lnk (G220V/R, A223V, G229S, and D234N) are in proximity to P317, R318, and P322 in SH2B1. In addition, eight of the human variants (E208Q, A215V, G220 V/R, G229S, D234N in SH2B3/Lnk and E299G, P322S in SH2B1) as well as P317 and R318 in SH2B1 are on or in proximity to this putative protein-binding interface ( Fig. 7 and Video 1).
The number of human variants in SH2B1 and SH2B3 in this region of the PH domain suggests that small structural changes in this region as a result of mutation or other modification have the potential to produce substantial functional consequences. Because the residues corresponding to P317 and R318 in SH2B1 are on the surface of the PH domain and do not substantially change the direction of the loop, the P317, R318 deletion in SH2B1 would shorten the loop but not severely damage the overall structure. However, the deletion would be expected to diminish stabilization of the turn provided by the predicted p-p stacking between residues P317 and F309 in SH2B1. In addition, the deletion would be expected to alter the shape and electrostatics of the interface surface in the region of P317 and R318 in SH2B1.
Because SH2B1 from humans and mice share 95% sequence identity, with only one conservative difference (S325T) near P322 (Fig. 7), the structures in mouse and human are expected to be nearly identical. Therefore, the more modest phenotypes of P322S/1 mice compared with humans may be due to differences in the affinity of PH domain binding partners. The mouse binding partners may be able to accommodate the P322S mutation better than human binding partners, and/or human physiology adapts more poorly to the resultant alterations in SH2B1 function. Given the different phenotypes produced by the P322S and DPR mutations in mice, we postulate that the two mutations alter the structure of the SH2B1 PH domain in different ways to produce distinct changes in cell physiology. That relatively small changes in the PH domain, predicted to have only minor effects on PH domain structure, cause a rather profound effect on SH2B1b localization at the cellular level and energy balance and glucose homeostasis at the whole-animal level provides some of the first real evidence of the importance of the PH domain in SH2B1 function. While SH2B1b has been shown to cycle through the nucleus, it is generally found at the plasma membrane and in the cytoplasm (22,23,26). The accumulation of SH2B1b DPR in the nucleus indicates that the DPR deletion greatly alters the ratio between nuclear import and nuclear export of SH2B1b. Consistently, the DPR mutation as well as many of the other human obesity-associated SH2B1 variants impair the ability of SH2B1b to promote neurotrophic factor-induced neurite outgrowth of PC12 cells. Because neurite outgrowth in PC12 cells shares many properties Video 1-This image is from a video available online at https://bcove .video/2lNxwhM. Modeling of the three-dimensional structure of the PH domain of SH2B1. A model of human SH2B1 was created by overlaying the sequence of the PH domain of human SH2B1 onto the mouse structure of APS. The two PH domain sequences are 75% similar (55.3% identical). The SH2B1 model is missing a single amino acid insertion at residue 296 and the 16-residue insertion at residue 262 which contains the R270W/Q human variant in SH2B1. The amino acid sequence of residues surrounding the P322 site is highly conserved between the two proteins. P322 is shown as yellow sticks. The other human variants in SH2B1 as well as residues P317 and R318 are magenta sticks. The human variants in Lnk are orange sticks. Differences between mouse and human SH2B1 are brown (no sticks). Oxygen atoms are red and nitrogen blue. b-Pleated sheets, a-helices, connecting loops, and hydrogen bonds in the region between the N-terminal end of b-strand 2 and the loop containing P317, R318, and P322 are indicated. The surface of the PH domain is tinted gray. P322 in SH2B1 is within 8 Å of the site of the D234N variant in SH2B3/ Lnk. The turn that contains D234N also contains the G229S variant in SH2B3/Lnk and the E299G variant in SH2B1. P317 and R318 in SH2B1 are within 8 Å of the site of G220 R/V and A223V in SH2B3/Lnk. This region is stabilized by p-p stacking between P317 and F309 in SH2B1 (P248 and F240 in Lnk) and a network of hydrogen bonds.
with the formation of axons and/or dendrites (50), and because the Sh2b1 KO mice have impaired leptin signaling, it will be important in the future to examine the impact of SH2B1 PH domain changes on the structure of neurons that control energy balance.