Interleukin-3 (IL-3) promotes both self-renewal and differentiation of early multipotential progenitors and is involved in inducible hematopoiesis in response to infections. Here we report new insights into these processes with the identification of a new isoform (SP2) of IL-3 receptor ␣ (IL-3R␣), present in mouse and human hematopoietic cells, which lacks domain 1 of the full-length receptor (SP1). Binding assays with ␤ IL-3 mutants showed that mouse SP2 uses a different high affinity binding mode to SP1, although both mouse and human SP2 and SP1 can stimulate IL-3-dependent growth. In IL-3-dependent differentiation models, human SP2 and SP1 gave differential effects on lineage commitment or self-renewal dependent on the cellular context, suggesting that different modes of ectodomain binding may modulate intracellular signaling. In a multipotential factor dependent cell-Paterson mix, the transcription factors C/EBP␣ and PU.1 and microRNAs miRNA-15a, -223, and -181a were up-regulated in cells undergoing SP2-supported differentiation compared with SP1-supported self-renewal. Similarly in M1 cells, SP2 promoted differentiation compared with SP1 and gave up-regulation of PU.1 and miRNA-155 and -223. These findings suggest that IL-3-promoted lineage commitment uses similar mechanisms to those of steady-state hematopoiesis. Both the SP1 and SP2 isoforms activated the Jak2/STAT5, Akt, and Erk1/2 signaling pathways in M1 cells, although the activation was more prolonged for the SP2 isoform.

Junctin, a non-catalytic splice variant encoded by the aspartate-β-hydroxylase (Asph) gene, is inserted into the membrane of the sarcoplasmic reticulum (SR) Ca2+ store where it modifies Ca2+ signalling in the heart and skeletal muscle through its regulation of ryanodine receptor (RyR) Ca2+ release channels. Junctin is required for normal muscle function as its knockout leads to abnormal Ca2+ signalling, muscle dysfunction and cardiac arrhythmia. However, the nature of the molecular interaction between junctin and RyRs is largely unknown and was assumed to occur only in the SR lumen. We find that there is substantial binding of RyRs to full junctin, and the junctin luminal and, unexpectedly, cytoplasmic domains. Binding of these different junctin domains had distinct effects on RyR1 and RyR2 activity: full junctin in the luminal solution increased RyR channel activity by ∼threefold, the C-terminal luminal interaction inhibited RyR channel activity by ∼50%, and the N-terminal cytoplasmic binding produced an ∼fivefold increase in RyR activity. The cytoplasmic interaction between junctin and RyR is required for luminal binding to replicate the influence of full junctin on RyR1 and RyR2 activity. The C-terminal domain of junctin binds to residues including the S1–S2 linker of RyR1 and N-terminal domain of junctin binds between RyR1 residues 1078 and 2156.

The contractile function of the heart requires the release of Ca(2+) from intracellular Ca(2+) stores in the sarcoplasmic reticulum (SR) of cardiac muscle cells. The efficacy of Ca(2+) release depends on the amount of Ca(2+) loaded into the Ca(2+) store and the way in which this 'Ca(2+) load' influences the activity of the cardiac ryanodine receptor Ca(2+) release channel (RyR2). The effects of the Ca(2+) load on Ca(2+) release through RyR2 are facilitated by: (i) the sensitivity of RyR2 itself to luminal Ca(2+) concentrations; and (ii) interactions between the cardiac Ca(2+) -binding protein calsequestrin (CSQ) 2 and RyR2, transmitted through the 'anchoring' proteins junctin and/or triadin. Mutations in RyR2 are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT) and sudden cardiac death. The tachycardia is associated with changes in the sensitivity of RyR2 to luminal Ca(2+) . Triadin-, junctin- or CSQ-null animals survive, but their longevity and ability to tolerate stress is compromised. These studies reveal the importance of the proteins in normal muscle function, but do not reveal the molecular nature of their functional interactions, which must be defined before changes in the proteins leading to CPVT and heart disease can be understood. Herein, we discuss known interactions between the RyR, triadin, junctin and CSQ with emphasis on the cardiac isoforms of the proteins. Where there is little known about the cardiac isoforms, we discuss evidence from skeletal isoforms.

GM-CSF (granulocyte/macrophage colony-stimulating factor) is an important mediator of inducible haemopoiesis and inflammation, and has a critical role in the function of alveolar macrophages. Its clinical applications include the mobilization of haemopoietic progenitors, and a role as an immune stimulant and vaccine adjuvant in cancer patients. GM-CSF signals via a specific alpha receptor (GM-CSFRalpha) and the shared hbetac (human common beta-subunit). The present study has investigated the role of the Ig-like domain of GM-CSFRalpha in GM-CSF binding and signalling. Deletion of the Ig-like domain abolished direct GM-CSF binding and decreased growth signalling in the presence of hbetac. To locate the specific residues in the Ig-like domain of GM-CSFRalpha involved in GM-CSF binding, a structural alignment was made with a related receptor, IL-13Ralpha1 (interleukin-13 receptor alpha1), whose structure and mode of interaction with its ligand has recently been elucidated. Mutagenesis of candidate residues in the predicted region of interaction identified Val51 and Cys60 as having critical roles in binding to the alpha receptor, with Arg54 and Leu55 also being important. High-affinity binding in the presence of hbetac was strongly affected by mutation of Cys60 and was also reduced by mutation of Val51, Arg54 and Leu55. Of the four key residues, growth signalling was most severely affected by mutation of Cys60. The results indicate a previously unrecognized role for the Ig-like domain, and in particular Cys60, of GM-CSFRalpha in the binding of GM-CSF and subsequent activation of cellular signalling.

The second of three SPRY domains (SPRY2, S1085 -V1208) located in the skeletal muscle ryanodine receptor (RyR1) is contained within regions of RyR1 that influence EC coupling and bind to imperatoxin A, a toxin probe of RyR1 channel gating. We examined the binding of the F loop (P1107-A1121) in SPRY2 to the ASI/basic region in RyR1 (T3471-G3500, containing both alternatively spliced (ASI) residues and neighboring basic amino acids). We then investigated the possible influence of this interaction on excitation contraction (EC) coupling. A peptide with the F loop sequence and an antibody to the SPRY2 domain each enhanced RyR1 activity at low concentrations and inhibited at higher concentrations. A peptide containing the ASI/basic sequence bound to SPRY2 and binding decreased ~10-fold following mutation or structural disruption of the basic residues. Binding was abolished by mutation of three critical acidic F loop residues. Together these results suggest that the ASI/basic and SPRY2 domains interact in an F loop regulatory module. Although a region that includes the SPRY2 domain influences EC coupling, as does the ASI/basic region, Ca2+ release during ligand- and depolarization-induced RyR1 activation were not altered by mutation of the three critical F loop residues following expression of mutant RyR1 in RyR1-null myotubes. Therefore the electrostatic regulatory interaction between the SPRY2 F loop residues (that bind to imperatoxin A) and the ASI/basic residues of RyR1 does not influence bi-directional DHPR-RyR1 signaling during skeletal EC coupling, possibly because the interaction is interrupted by the influence of factors present in intact muscle cells.