Cell and Tissue Research (2020) 382:57–64 https://doi.org/10.1007/s00441-020-03235-4 AT-A-GLANCE ARTICLE Plasma membrane localization of the GFL receptor components: a nexus for receptor crosstalk Christopher R. Donnelly1 & Brian A. Pierchala2 Received: 19 March 2020 /Accepted: 4 June 2020 / Published online: 7 August 2020 # The Author(s) 2020 Abstract The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) comprise a group of four homologous and potent growth factors that includes GDNF, neurturin (NRTN), artemin (ARTN), and persephin (PSPN). The survival, growth, and mitotic activities of the GFLs are conveyed by a single receptor tyrosine kinase, Ret. The GFLs do not bind directly to Ret in order to activate it, and instead bind with high affinity to glycerophosphatidylinositol (GPI)-anchored coreceptors called the GDNF family receptor-αs(GFRαs). Several mechanisms have recently been identified that influence the trafficking of Ret and GFRαs in and out of the plasma membrane, thereby affecting their availability for ligand binding, as well as their levels by targeting to degradative pathways. This review describes these mechanisms and their powerful effects on GFL signaling and function. We also describe the recent discovery that p75 and Ret form a signaling complex, also regulated by plasma membrane shuttling, that either enhances GFL survival signals or p75 pro-apoptotic signals, dependent on the cellular context. Keywords Ret . GFRα . Plasma membrane . Intracellular trafficking . GDNF . GFLs . TGF-β regulation Introduction receptors, as well as other classes of receptors, and the cellular context of these interactions ultimately determines the fate of In the past, illustrations detailing neurotrophic factor signaling neurons during neural development (e.g., survival, death, dif- pathways often indicated relatively simplistic interactions be- ferentiation, axon growth). Interactions between neurotrophic tween well-established ligand-receptor pairs, with each com- factor receptor signaling pathways have been demonstrated in plex separately leading to the activation of downstream sig- a variety of cellular contexts, including (1) indirectly, via in- naling cascades controlling a variety of cell fates. Within the tegration of shared downstream signaling pathways; (2) di- neurotrophin family, for example, nerve growth factor (NGF) rectly, through ligand-dependent or ligand-independent inter- binds to TrkA, brain-derived neurotrophic factor (BDNF) and actions at the cell surface; and (3) through mechanisms in- neurotrophin-4 (NT-4) bind to TrkB, and neurotrophin-3 volving receptor sorting, trafficking, or membrane binds to TrkC, and all four ligands can bind with reduced localization. Here we provide a brief overview of neurotrophic affinity to p75 (Reichardt 2006). However, as our understand- factor receptor crosstalk with an emphasis on Ret, the receptor ing of neurotrophic factor signaling has evolved over the last tyrosine kinase for the glial cell line-derived neurotrophic fac- several decades, we have come to appreciate that a highly tor (GDNF) family of ligands (GFLs), for which all three of complex set of interactions exists between neurotrophic factor these mechanisms has recently come to light. The GFLs are a family of neurotrophic factors distantly related to the transforming growth factor-β (TGF-β)super- family, consisting of four homologous members: GDNF, * Brian A. Pierchala [email protected] neurturin (NRTN), artemin (ARTN), and persephin (PSPN). The GFLs exert their functions by first binding as homodimers 1 Department of Anesthesiology Center for Translational Pain to lipid raft–localized glycophosphatidylinositol (GPI)-linked Medicine, Duke University Medical Center, Durham, NC 27710, co-receptors known as the GDNF family receptor-αs USA (GFRαs), which then recruit and engage Ret. Each GFL has 2 Department of Anatomy, Cell Biology & Physiology, Stark a preferred GFRα co-receptor, with GDNF preferentially Neurosciences Research Institute, Indiana University School of binding to GFRα1, NRTN to GFRα2, ARTN to GFRα3, Medicine, Indianapolis, IN 46202, USA 58 Cell Tissue Res (2020) 382:57–64 and PSPN to GFRα4, although cross-binding can occur Interestingly, the neuroprotective effect of GDNF is abolished (Airaksinen and Saarma 2002). Of note, GFL signaling is by the co-treatment of TGF-β neutralizing antibodies with unique: Ret remains the only RTK identified to date which GDNF (Schober et al. 1999). Consistent with these models, does not bind its ligands directly but instead requires a co- co-application of GDNF and TGF-β promotes the survival of receptor for activation. This peculiarity thus confers additional developing sympathetic neurons of the chicken ciliary gangli- opportunities to fine-tune GFL signaling through dynamic on (CG), while neither factor on their own is able to promote regulation of both Ret and GFRαs. Since the initial discovery survival (Peterziel et al. 2002). Of note, these effects are of GDNF-Ret signaling as a critical regulator of kidney and blocked by extracellular signal-regulated kinase (ERK) inhi- enteric nervous system development in 1996 (Pichel et al. bition, but not by inhibition of the PI3 kinase (PI3K) pathway 1996; Sanchez et al. 1996;Schuchardtetal.1994), the bio- (Peterziel et al. 2002). While CG neurons are unresponsive to logical functions of Ret have been greatly expanded. In the physiological concentrations of GDNF at baseline, TGF-β nervous system, GFL-Ret signaling is now appreciated to play pretreatment for 3 h is sufficient to induce GDNF responsive- a critical role in the development of several populations of ness without requiring TGF-β to continue to be present along dorsal root ganglion (DRG) somatosensory neurons (Luo with GDNF. In fact, after pretreatment with TGF-β, the com- et al. 2007, 2009; Molliver et al. 1997)aswellasgeniculate plete elimination of TGF-β using function-blocking antibod- oral sensory neurons (Donnelly et al. 2017), spinal motor neu- ies in combination with GDNF does not alter the survival rons (Dudanova et al. 2010; Gould et al. 2008; Haase et al. effects of GDNF (Peterziel et al. 2002). Mechanistically, 2002), postganglionic sympathetic and parasympathetic neu- TGF-β induces the cell surface localization of GFRα1with- rons (Durbec et al. 1996; Enomoto et al. 2001; Enomoto et al. out affecting either GFRα1 mRNA or protein levels (Peterziel 2000), orofacial trigeminal nociceptors (Donnelly et al. 2019), et al. 2002). NRTN, in contrast, was found to promote the and even other populations as well. Ret has recently been survival of developing CG neurons in the absence of found to be activated by growth differentiation factor 15 TGF-β, and disrupting TGF-β signaling using a pharmaco- (GDF15), a metabolic regulator that binds to the co-receptor logical inhibitor of TGF-β Receptor 2 (Tgfrb2) failed to at- GFRAL and together engages Ret (Mullican et al. 2017; Yang tenuate NRTN-mediated survival (Peterziel et al. 2007). et al. 2017). Whether Ret is the predominant signal- Consistent with these data, TGF-β induced the recruitment transducing receptor component underlying the effects of of GFRα1 to the cell surface but did not recruit GFRα2, GDF15 on food intake and obesity is under investigation. which was already abundantly localized to the cell surface Beyond normal physiological function, aberrant Ret signaling (Peterziel et al. 2007). Taken together, TGF-β is uniquely is also implicated in numerous cancers (Plaza-Menacho et al. involved in GDNF signal transduction via a TGF-β/Tgfrb2/ 2014). Thus, an appreciation of the regulatory mechanisms MAPK pathway that promotes the trafficking of GFRα1to governing GFL-Ret signal transduction has the capacity to the plasma membrane (Fig. 1). To date, it remains unknown inform our understanding of a variety of different develop- whether TGF-β affects localization of the other two GFL re- mental and disease processes. ceptor complexes (e.g., Ret, GFRα3, GFRα4) and mechanis- tically how TGF-β-mediated MAPK activation triggers the TGF-β regulation of GFL signaling through GFRα1 movement of internal stores of GFRα1 to the cell surface. surface localization Shutting off the signal: GDNF, GFRα1, and Ret Given the important biological functions carried out by the trafficking by SorLA GFLs, it is perhaps not surprising that several mechanisms exist to precisely regulate GFL signaling through cell surface Although considerable progress has been made in our under- routing and membrane localization of the GFL receptor com- standing of GFL receptor signaling and its downstream con- ponents. Interestingly, TGF-β has an important role in the sequences, less is known about how ongoing GDNF-GFRα1- trophic functions of GDNF in developing neurons, both Ret signaling is extinguished at the molecular level. On this in vitro and in vivo. Krieglstein et al. demonstrated that note, the sorting receptor SorLA was recently demonstrated to GDNF-mediated survival of several developing peripheral have a critical role. Using heterologous cell lines, it has been and central neuron populations in vitro required the addition reported that the proteolytic processing of proGDNF to mature of TGF-β, and moreover, TGF-β neutralizing antibodies GDNF that occurs during transit through the secretory path- could abolish GDNF-mediated neuronal survival way requires SorLA (Geng et al. 2011). SorLA binds directly (Krieglstein et al. 1998). In an in vivo model of to both proGDNF via the prodomain and to mature GDNF, adrenomedullectomy,