Enhanced sodium channel inactivation by temperature and FHF2 deficiency blocks heat nociception.

ABSTRACT: Transient voltage-gated sodium currents are essential for the initiation and conduction of action potentials in neurons and cardiomyocytes. The amplitude and duration of sodium currents are tuned by intracellular fibroblast growth factor homologous factors (FHFs/iFGFs) that associate with the cytoplasmic tails of voltage-gated sodium channels (Na v s), and genetic ablation of Fhf genes disturbs neurological and cardiac functions. Among reported phenotypes, Fhf2null mice undergo lethal hyperthermia-induced cardiac conduction block attributable to the combined effects of FHF2 deficiency and elevated temperature on the cardiac sodium channel (Na v 1.5) inactivation rate. Fhf2null mice also display a lack of heat nociception, while retaining other somatosensory capabilities. Here, we use electrophysiological and computational methods to show that the heat nociception deficit can be explained by the combined effects of elevated temperature and FHF2 deficiency on the fast inactivation gating of Na v 1.7 and tetrodotoxin-resistant sodium channels expressed in dorsal root ganglion C fibers. Hence, neurological and cardiac heat-associated deficits in Fhf2null mice derive from shared impacts of FHF deficiency and temperature towards Na v inactivation gating kinetics in distinct tissues.

Early onset epilepsy and sudden unexpected death in epilepsy with cardiac arrhythmia in mice carrying the early infantile epileptic encephalopathy 47 gain-of-function FHF1(FGF12) missense mutation.

OBJECTIVE: Fibroblast growth factor homologous factors (FHFs) are brain and cardiac sodium channel-binding proteins that modulate channel density and inactivation gating. A recurrent de novo gain-of-function missense mutation in the FHF1(FGF12) gene (p.Arg52His) is associated with early infantile epileptic encephalopathy 47 (EIEE47; Online Mendelian Inheritance in Man database 617166). To determine whether the FHF1 missense mutation is sufficient to cause EIEE and to establish an animal model for EIEE47, we sought to engineer this mutation into mice. METHODS: The Arg52His mutation was introduced into fertilized eggs by CRISPR (clustered regularly interspaced short palindromic repeats) editing to generate Fhf1R52H/F+ mice. Spontaneous epileptiform events in Fhf1R52H/+ mice were assessed by cortical electroencephalography (EEG) and video monitoring. Basal heart rhythm and seizure-induced arrhythmia were recorded by electrocardiography. Modulation of cardiac sodium channel inactivation by FHF1BR52H protein was assayed by voltage-clamp recordings of FHF-deficient mouse cardiomyocytes infected with adenoviruses expressing wild-type FHF1B or FHF1BR52H protein. RESULTS: All Fhf1R52H/+ mice experienced seizure or seizurelike episodes with lethal ending between 12 and 26 days of age. EEG recordings in 19-20-day-old mice confirmed sudden unexpected death in epilepsy (SUDEP) as severe tonic seizures immediately preceding loss of brain activity and death. Within 2-53 s after lethal seizure onset, heart rate abruptly declined from 572 ± 16 bpm to 108 ± 15 bpm, suggesting a parasympathetic surge accompanying seizures that may have contributed to SUDEP. Although ectopic overexpression of FHF1BR52H in cardiomyocytes induced a 15-mV depolarizing shift in voltage of steady-state sodium channel inactivation and slowed the rate of channel inactivation, heart rhythm was normal in Fhf1R52H/+ mice prior to seizure. SIGNIFICANCE: The Fhf1 missense mutation p.Arg52His induces epileptic encephalopathy with full penetrance in mice. Both Fhf1 (p.Arg52His) and Scn8a (p.Asn1768Asp) missense mutations enhance sodium channel Nav 1.6 currents and induce SUDEP with bradycardia in mice, suggesting an FHF1/Nav 1.6 functional axis underlying altered brain sodium channel gating in epileptic encephalopathy.

Missense variants in the N-terminal domain of the A isoform of FHF2/FGF13 cause an X-linked developmental and epileptic encephalopathy.

Fibroblast growth factor homologous factors (FHFs) are intracellular proteins which regulate voltage-gated sodium (Nav) channels in the brain and other tissues. FHF dysfunction has been linked to neurological disorders including epilepsy. Here, we describe two sibling pairs and three unrelated males who presented in infancy with intractable focal seizures and severe developmental delay. Whole-exome sequencing identified hemi- and heterozygous variants in the N-terminal domain of the A isoform of FHF2 (FHF2A). The X-linked FHF2 gene (also known as FGF13) has alternative first exons which produce multiple protein isoforms that differ in their N-terminal sequence. The variants were located at highly conserved residues in the FHF2A inactivation particle that competes with the intrinsic fast inactivation mechanism of Nav channels. Functional characterization of mutant FHF2A co-expressed with wild-type Nav1.6 (SCN8A) revealed that mutant FHF2A proteins lost the ability to induce rapid-onset, long-term blockade of the channel while retaining pro-excitatory properties. These gain-of-function effects are likely to increase neuronal excitability consistent with the epileptic potential of FHF2 variants. Our findings demonstrate that FHF2 variants are a cause of infantile-onset developmental and epileptic encephalopathy and underline the critical role of the FHF2A isoform in regulating Nav channel function.

Ionic Mechanisms of Impulse Propagation Failure in the FHF2-Deficient Heart.

RATIONALE: FHFs (fibroblast growth factor homologous factors) are key regulators of sodium channel (NaV) inactivation. Mutations in these critical proteins have been implicated in human diseases including Brugada syndrome, idiopathic ventricular arrhythmias, and epileptic encephalopathy. The underlying ionic mechanisms by which reduced Nav availability in Fhf2 knockout (Fhf2KO) mice predisposes to abnormal excitability at the tissue level are not well defined. OBJECTIVE: Using animal models and theoretical multicellular linear strands, we examined how FHF2 orchestrates the interdependency of sodium, calcium, and gap junctional conductances to safeguard cardiac conduction. METHODS AND RESULTS: Fhf2KO mice were challenged by reducing calcium conductance (gCaV) using verapamil or by reducing gap junctional conductance (Gj) using carbenoxolone or by backcrossing into a cardiomyocyte-specific Cx43 (connexin 43) heterozygous background. All conditions produced conduction block in Fhf2KO mice, with Fhf2 wild-type (Fhf2WT) mice showing normal impulse propagation. To explore the ionic mechanisms of block in Fhf2KO hearts, multicellular linear strand models incorporating FHF2-deficient Nav inactivation properties were constructed and faithfully recapitulated conduction abnormalities seen in mutant hearts. The mechanisms of conduction block in mutant strands with reduced gCaV or diminished Gj are very different. Enhanced Nav inactivation due to FHF2 deficiency shifts dependence onto calcium current (ICa) to sustain electrotonic driving force, axial current flow, and action potential (AP) generation from cell-to-cell. In the setting of diminished Gj, slower charging time from upstream cells conspires with accelerated Nav inactivation in mutant strands to prevent sufficient downstream cell charging for AP propagation. CONCLUSIONS: FHF2-dependent effects on Nav inactivation ensure adequate sodium current (INa) reserve to safeguard against numerous threats to reliable cardiac impulse propagation.

Hyperexcitability and Hyperplasticity Disrupt Cerebellar Signal Transfer in the IB2 KO Mouse Model of Autism.

Autism spectrum disorders (ASDs) are pervasive neurodevelopmental conditions that often involve mutations affecting synaptic mechanisms. Recently, the involvement of cerebellum in ASDs has been suggested, but the underlying functional alterations remained obscure. We investigated single-neuron and microcircuit properties in IB2 (Islet Brain-2) KO mice of either sex. The IB2 gene (chr22q13.3 terminal region) deletion occurs in virtually all cases of Phelan-McDermid syndrome, causing autistic symptoms and a severe delay in motor skill acquisition. IB2 KO granule cells showed a larger NMDA receptor-mediated current and enhanced intrinsic excitability, raising the excitatory/inhibitory balance. Furthermore, the spatial organization of granular layer responses to mossy fibers shifted from a "Mexican hat" to a "stovepipe hat" profile, with stronger excitation in the core and weaker inhibition in the surround. Finally, the size and extension of long-term synaptic plasticity were remarkably increased. These results show for the first time that hyperexcitability and hyperplasticity disrupt signal transfer in the granular layer of IB2 KO mice, supporting cerebellar involvement in the pathogenesis of ASD.SIGNIFICANCE STATEMENT This article shows for the first time a complex set of alterations in the cerebellum granular layer of a mouse model [IB2 (Islet Brain-2) KO] of autism spectrum disorders. The IB2 KO in mice mimics the deletion of the corresponding gene in the Phelan-McDermid syndrome in humans. The changes reported here are centered on NMDA receptor hyperactivity, hyperplasticity, and hyperexcitability. These, in turn, increase the excitatory/inhibitory balance and alter the shape of center/surround structures that emerge in the granular layer in response to mossy fiber activity. These results support recent theories suggesting the involvement of cerebellum in autism spectrum disorders.

FGF-Dependent, Context-Driven Role for FRS Adapters in the Early Telencephalon.

FGF signaling, an important component of intercellular communication, is required in many tissues throughout development to promote diverse cellular processes. Whether FGF receptors (FGFRs) accomplish such varied tasks in part by activating different intracellular transducers in different contexts remains unclear. Here, we used the developing mouse telencephalon as an example to study the role of the FRS adapters FRS2 and FRS3 in mediating the functions of FGFRs. Using tissue-specific and germline mutants, we examined the requirement of Frs genes in two FGFR-dependent processes. We found that Frs2 and Frs3 are together required for the differentiation of a subset of medial ganglionic eminence (MGE)-derived neurons, but are dispensable for the survival of early telencephalic precursor cells, in which any one of three FGFRs (FGFR1, FGFR2, or FGFR3) is sufficient for survival. Although FRS adapters are dispensable for ERK-1/2 activation, they are required for AKT activation within the subventricular zone of the developing MGE. Using an FRS2,3-binding site mutant of Fgfr1, we established that FRS adapters are necessary for mediating most or all FGFR1 signaling, not only in MGE differentiation, but also in cell survival, implying that other adapters mediate at least in part the signaling from FGFR2 and FGFR3. Our study provides an example of a contextual role for an intracellular transducer and contributes to our understanding of how FGF signaling plays diverse developmental roles.SIGNIFICANCE STATEMENT FGFs promote a range of developmental processes in many developing tissues and at multiple developmental stages. The mechanisms underlying this multifunctionality remain poorly defined in vivo Using telencephalon development as an example, we show here that FRS adapters exhibit some selectivity in their requirement for mediating FGF receptor (FGFR) signaling and activating downstream mediators that depend on the developmental process, with a requirement in neuronal differentiation but not cell survival. Differential engagement of FRS and non-FRS intracellular adapters downstream of FGFRs could therefore in principle explain how FGFs play several distinct roles in other developing tissues and developmental stages.

Fhf2 gene deletion causes temperature-sensitive cardiac conduction failure.

Fever is a highly conserved systemic response to infection dating back over 600 million years. Although conferring a survival benefit, fever can negatively impact the function of excitable tissues, such as the heart, producing cardiac arrhythmias. Here we show that mice lacking fibroblast growth factor homologous factor 2 (FHF2) have normal cardiac rhythm at baseline, but increasing core body temperature by as little as 3 °C causes coved-type ST elevations and progressive conduction failure that is fully reversible upon return to normothermia. FHF2-deficient cardiomyocytes generate action potentials upon current injection at 25 °C but are unexcitable at 40 °C. The absence of FHF2 accelerates the rate of closed-state and open-state sodium channel inactivation, which synergizes with temperature-dependent enhancement of inactivation rate to severely suppress cardiac sodium currents at elevated temperatures. Our experimental and computational results identify an essential role for FHF2 in dictating myocardial excitability and conduction that safeguards against temperature-sensitive conduction failure.

FHF-independent conduction of action potentials along the leak-resistant cerebellar granule cell axon.

Neurons in vertebrate central nervous systems initiate and conduct sodium action potentials in distinct subcellular compartments that differ architecturally and electrically. Here, we report several unanticipated passive and active properties of the cerebellar granule cell's unmyelinated axon. Whereas spike initiation at the axon initial segment relies on sodium channel (Nav)-associated fibroblast growth factor homologous factor (FHF) proteins to delay Nav inactivation, distal axonal Navs show little FHF association or FHF requirement for high-frequency transmission, velocity and waveforms of conducting action potentials. In addition, leak conductance density along the distal axon is estimated as <1% that of somatodendritic membrane. The faster inactivation rate of FHF-free Navs together with very low axonal leak conductance serves to minimize ionic fluxes and energetic demand during repetitive spike conduction and at rest. The absence of FHFs from Navs at nodes of Ranvier in the central nervous system suggests a similar mechanism of current flux minimization along myelinated axons.

Gain-of-function FHF1 mutation causes early-onset epileptic encephalopathy with cerebellar atrophy.

OBJECTIVE: Voltage-gated sodium channel (Nav)-encoding genes are among early-onset epileptic encephalopathies (EOEE) targets, suggesting that other genes encoding Nav-binding proteins, such as fibroblast growth factor homologous factors (FHFs), may also play roles in these disorders. METHODS: To identify additional genes for EOEE, we performed whole-exome sequencing in a family quintet with 2 siblings with a lethal disease characterized by EOEE and cerebellar atrophy. The pathogenic nature and functional consequences of the identified sequence alteration were determined by electrophysiologic studies in vitro and in vivo. RESULTS: A de novo heterozygous missense mutation was identified in the FHF1 gene (FHF1AR114H, FHF1BR52H) in the 2 affected siblings. The mutant FHF1 proteins had a strong gain-of-function phenotype in transfected Neuro2A cells, enhancing the depolarizing shifts in Nav1.6 voltage-dependent fast inactivation, predicting increased neuronal excitability. Surprisingly, the gain-of-function effect is predicted to result from weaker interaction of mutant FHF1 with the Nav cytoplasmic tail. Transgenic overexpression of mutant FHF1B in zebrafish larvae enhanced epileptiform discharges, demonstrating the epileptic potential of this FHF1 mutation in the affected children. CONCLUSIONS: Our data demonstrate that gain-of-function FHF mutations can cause neurologic disorder, and expand the repertoire of genetic causes (FHF1) and mechanisms (altered Nav gating) underlying EOEE and cerebellar atrophy.

Fast-onset long-term open-state block of sodium channels by A-type FHFs mediates classical spike accommodation in hippocampal pyramidal neurons.

Classical accommodation is a form of spike frequency adaptation in neurons whereby excitatory drive results in action potential output of gradually decreasing frequency. Here we describe an essential molecular component underlying classical accommodation in juvenile mouse hippocampal CA1 pyramidal neurons. A-type isoforms of fibroblast growth factor homologous factors (FHFs) bound to axosomatic voltage-gated sodium channels bear an N-terminal blocking particle that drives some associated channels into a fast-onset, long-term inactivated state. Use-dependent accumulating channel blockade progressively elevates spike voltage threshold and lengthens interspike intervals. The FHF particle only blocks sodium channels from the open state, and mutagenesis studies demonstrate that this particle uses multiple aliphatic and cationic residues to both induce and maintain the long-term inactivated state. The broad expression of A-type FHFs in neurons throughout the vertebrate CNS suggests a widespread role of these sodium channel modulators in the control of neural firing.

Voltage-gated sodium channel-associated proteins and alternative mechanisms of inactivation and block.

Voltage-gated sodium channels mediate inward current of action potentials upon membrane depolarization of excitable cells. The initial transient sodium current is restricted to milliseconds through three distinct channel-inactivating and blocking mechanisms. All pore-forming alpha subunits of sodium channels possess structural elements mediating fast inactivation upon depolarization and recovery within milliseconds upon membrane repolarization. Accessory subunits modulate fast inactivation dynamics, but these proteins can also limit current by contributing distinct inactivation and blocking particles. A-type isoforms of fibroblast growth factor homologous factors (FHFs) bear a particle that induces long-term channel inactivation, while sodium channel subunit Navß4 employs a blocking particle that rapidly dissociates upon membrane repolarization to generate resurgent current. Despite their different physiological functions, the FHF and Navß4 particles have similarity in amino acid composition and mechanisms for docking within sodium channels. The three competing channel-inactivating and blocking processes functionally interact to regulate a neuron's intrinsic excitability.

Behavioral and cerebellar transmission deficits in mice lacking the autism-linked gene islet brain-2.

Deletion of the human SHANK3 gene near the terminus of chromosome 22q is associated with Phelan-McDermid syndrome and autism spectrum disorders. Nearly all such deletions also span the tightly linked IB2 gene. We show here that IB2 protein is broadly expressed in the brain and is highly enriched within postsynaptic densities. Experimental disruption of the IB2 gene in mice reduces AMPA and enhances NMDA receptor-mediated glutamatergic transmission in cerebellum, changes the morphology of Purkinje cell dendritic arbors, and induces motor and cognitive deficits suggesting an autism phenotype. These findings support a role for human IB2 mutation as a contributing genetic factor in Chr22qter-associated cognitive disorders.

Long-term inactivation particle for voltage-gated sodium channels.

Action potential generation is governed by the opening, inactivation, and recovery of voltage-gated sodium channels. A channel's voltage-sensing and pore-forming α subunit bears an intrinsic fast inactivation particle that mediates both onset of inactivation upon membrane depolarization and rapid recovery upon repolarization. We describe here a novel inactivation particle housed within an accessory channel subunit (A-type FHF protein) that mediates rapid-onset, long-term inactivation of several sodium channels. The channel-intrinsic and tethered FHF-derived particles, both situated at the cytoplasmic face of the plasma membrane, compete for induction of inactivation, causing channels to progressively accumulate into the long-term refractory state during multiple cycles of membrane depolarization. Intracellular injection of a short peptide corresponding to the FHF particle can reproduce channel long-term inactivation in a dose-dependent manner and can inhibit repetitive firing of cerebellar granule neurons. We discuss potential structural mechanisms of long-term inactivation and potential roles of A-type FHFs in the modulation of action potential generation and conduction.

Crystal structure of a fibroblast growth factor homologous factor (FHF) defines a conserved surface on FHFs for binding and modulation of voltage-gated sodium channels.

Voltage-gated sodium channels (Nav) produce sodium currents that underlie the initiation and propagation of action potentials in nerve and muscle cells. Fibroblast growth factor homologous factors (FHFs) bind to the intracellular C-terminal region of the Nav alpha subunit to modulate fast inactivation of the channel. In this study we solved the crystal structure of a 149-residue-long fragment of human FHF2A which unveils the structural features of the homology core domain of all 10 human FHF isoforms. Through analysis of crystal packing contacts and site-directed mutagenesis experiments we identified a conserved surface on the FHF core domain that mediates channel binding in vitro and in vivo. Mutations at this channel binding surface impaired the ability of FHFs to co-localize with Navs at the axon initial segment of hippocampal neurons. The mutations also disabled FHF modulation of voltage-dependent fast inactivation of sodium channels in neuronal cells. Based on our data, we propose that FHFs constitute auxiliary subunits for Navs.

Axonal Na+ channels ensure fast spike activation and back-propagation in cerebellar granule cells.

In most neurons, Na+ channels in the axon are complemented by others localized in the soma and dendrites to ensure spike back-propagation. However, cerebellar granule cells are neurons with simplified architecture in which the dendrites are short and unbranched and a single thin ascending axon travels toward the molecular layer before bifurcating into parallel fibers. Here we show that in cerebellar granule cells, Na+ channels are enriched in the axon, especially in the hillock, but almost absent from soma and dendrites. The impact of this channel distribution on neuronal electroresponsiveness was investigated by multi-compartmental modeling. Numerical simulations indicated that granule cells have a compact electrotonic structure allowing excitatory postsynaptic potentials to diffuse with little attenuation from dendrites to axon. The spike arose almost simultaneously along the whole axonal ascending branch and invaded the hillock the activation of which promoted spike back-propagation with marginal delay (<200 micros) and attenuation (<20 mV) into the somato-dendritic compartment. These properties allow granule cells to perform sub-millisecond coincidence detection of pre- and postsynaptic activity and to rapidly activate Purkinje cells contacted by the axonal ascending branch.

Fibroblast growth factor homologous factors control neuronal excitability through modulation of voltage-gated sodium channels.

Neurons integrate and encode complex synaptic inputs into action potential outputs through a process termed "intrinsic excitability." Here, we report the essential contribution of fibroblast growth factor homologous factors (FHFs), a family of voltage-gated sodium channel binding proteins, to this process. Fhf1-/-Fhf4-/- mice suffer from severe ataxia and other neurological deficits. In mouse cerebellar slice recordings, WT granule neurons can be induced to fire action potentials repetitively (approximately 60 Hz), whereas Fhf1-/-Fhf4-/- neurons often fire only once and at an elevated voltage spike threshold. Sodium channels in Fhf1-/-Fhf4-/- granule neurons inactivate at more negative membrane potential, inactivate more rapidly, and are slower to recover from the inactivated state. Altered sodium channel physiology is sufficient to explain excitation deficits, as tested in a granule cell computer model. These findings offer a physiological mechanism underlying human spinocerebellar ataxia induced by Fhf4 mutation and suggest a broad role for FHFs in the control of excitability throughout the CNS.

Fibroblast growth factor homologous factors: evolution, structure, and function.

Fibroblast growth factor homologous factors (FHFs) bear strong sequence and structural similarity to fibroblast growth factors (FGFs). However, the biochemical and functional properties of FHFs are largely, if not totally, unrelated to those of FGFs. Whereas FGFs function through binding to the extracellular domains of FGF receptors (FGFRs), FHFs bind to intracellular domains of voltage-gated sodium channels (VGSCs) and to a neuronal MAP kinase scaffold protein, islet-brain-2 (IB2). These findings demonstrate the remarkable functional adaptability during evolution of the FGF gene family. FHF gene mutations in mice result in a range of neurological abnormalities, and at least one of these anomalies, cerebellar ataxia, is linked to FHF mutations in humans. This article reviews the sequences and structure of FHFs, along with our still limited understanding of FHF function.

Fibroblast growth factor homologous factor 2B: association with Nav1.6 and selective colocalization at nodes of Ranvier of dorsal root axons.

Voltage-gated sodium channels interact with cytosolic proteins that regulate channel trafficking and/or modulate the biophysical properties of the channels. Na(v)1.6 is heavily expressed at the nodes of Ranvier along adult CNS and PNS axons and along unmyelinated fibers in the PNS. In an initial yeast two-hybrid screen using the C terminus of Na(v)1.6 as a bait, we identified FHF2B, a member of the FGF homologous factor (FHF) subfamily, as an interacting partner of Na(v)1.6. Members of the FHF subfamily share approximately 70% sequence identity, and individual members demonstrate a cell- and tissue-specific expression pattern. FHF2 is abundantly expressed in the hippocampus and DRG neurons and colocalizes with Na(v)1.6 at mature nodes of Ranvier in myelinated sensory fibers in the dorsal root of the sciatic nerve. However, retinal ganglion cells and spinal ventral horn motor neurons show very low levels of FHF2 expression, and their axons exhibit no nodal FHF2 staining within the optic nerve and ventral root, respectively. Thus, FHF2 is selectively localized at nodes of dorsal root sensory but not ventral root motor axons. The coexpression of FHF2B and Na(v)1.6 in the DRG-derived cell line ND7/23 significantly increases the peak current amplitude and causes a 4 mV depolarizing shift of voltage-dependent inactivation of the channel. The preferential expression of FHF2B in sensory neurons may provide a basis for physiological differences in sodium currents that have been reported at the nodes of Ranvier in sensory versus motor axons.

Fibroblast growth factor (FGF) homologous factors share structural but not functional homology with FGFs.

Fibroblast growth factors (FGFs) interact with heparan sulfate glycosaminoglycans and the extracellular domains of FGF cell surface receptors (FGFRs) to trigger receptor activation and biological responses. FGF homologous factors (FHF1-FHF4; also known as FGF11-FGF14) are related to FGFs by substantial sequence homology, yet their only documented interactions are with an intracellular kinase scaffold protein, islet brain-2 (IB2) and with voltage-gated sodium channels. In this report, we show that recombinant FHFs can bind heparin with high affinity like classical FGFs yet fail to activate any of the seven principal FGFRs. Instead, we demonstrate that FHFs bind IB2 directly, furthering the contention that FHFs and FGFs elicit their biological effects by binding to different protein partners. To understand the molecular basis for this differential target binding specificity, we elucidated the crystal structure of FHF1b to 1.7-A resolution. The FHF1b core domain assumes a beta-trefoil fold consisting of 12 antiparallel beta strands (beta 1 through beta 12). The FHF1b beta-trefoil core is remarkably similar to that of classical FGFs and exhibits an FGF-characteristic heparin-binding surface as attested to by the number of bound sulfate ions. Using molecular modeling and structure-based mutational analysis, we identified two surface residues, Arg52 in the beta 4-beta 5 loop and Val95 in the beta 9 strand of FHF1b that are required for the interaction of FHF1b with IB2. These two residues are unique to FHFs, and mutations of the corresponding residues of FGF1 to Arg and Val diminish the capacity of FGF1 to activate FGFRs, suggesting that these two FHF residues contribute to the inability of FHFs to activate FGFRs. Hence, FHFs and FGFs bear striking structural similarity but have diverged to direct related surfaces toward interaction with distinct protein targets.