REEP1 and REEP2 proteins are preferentially expressed in neuronal and neuronal-like exocytotic tissues

Abstract

The six members of the Receptor Expression Enhancing Protein (REEP) family were originally identified based on their ability to enhance heterologous expression of olfactory receptors and other difficult to express G protein-coupled receptors. Interestingly, REEP1 mutations have been linked to neurodegenerative disorders of upper and lower motor neurons, hereditary spastic paraplegia (HSP) and distal hereditary motor neuropathy type V (dHMN-V). The closely related REEP2 isoform has not demonstrated any such disease linkage. Previous research has suggested that REEP1 mRNA is ubiquitously expressed in brain, muscle, endocrine, and multiple other organs, inconsistent with the neurodegenerative phenotype observed in HSP and dHMN-V. To more fully examine REEP1 expression, we developed and characterized a new REEP1 monoclonal antibody for both immunoblotting and immunofluorescent microscopic analysis. Unlike previous RT-PCR studies, immunoblotting demonstrated that REEP1 protein was not ubiquitous; its expression was restricted to neuronal tissues (brain, spinal cord) and testes. Gene expression microarray analysis demonstrated REEP1 and REEP2 mRNA expression in superior cervical and stellate sympathetic ganglia tissue. Furthermore, expression of endogenous REEP1 was confirmed in cultured murine sympathetic ganglion neurons by RT-PCR and immunofluorescent staining, with expression occurring between Day 4 and Day 8 of culture. Lastly, we demonstrated that REEP2 protein expression was also restricted to neuronal tissues (brain and spinal cord) and tissues that exhibit neuronal-like exocytosis (testes, pituitary, and adrenal gland). In addition to sensory tissues, expression of the REEP1/REEP2 subfamily appears to be restricted to neuronal and neuronal-like exocytotic tissues, consistent with neuronally restricted symptoms of REEP1 genetic disorders.

Keywords: AR; Distal hereditary motor neuropathy type V; G protein-coupled receptor; GPCR; HSP; Hereditary spastic paraplegia; NGF; Neurodegeneration; OR; REEP; RT-PCR; RTP; Receptor expression enhancing protein; SCG; SG; Sympathetic ganglion neuron; Yip; Ypt interacting protein; adrenergic receptor; dHMN-V; distal hereditary motor neuropathy type V; hereditary spastic paraplegia; mAb; monoclonal antibody; nerve growth factor; olfactory receptor; receptor expression enhancing protein; receptor transporting protein; reverse transcription polymerase chain reaction; stellate ganglion, SGN, sympathetic ganglion neurons; superior cervical ganglia.

Figure 1

Determination of REEP1 and REEP2 antisera specificity Whole cell lysates from various Flag-REEP1, -REEP2, -REEP6 transfected HEK293A cells (seventy-five micrograms protein/lane) were analyzed by immunoblot analysis with a new REEP1 mAb (NeuroMab Clone N345/51) or commercially available REEP2 polyclonal antisera to determine their REEP specificity. A. The REEP1 mAb detected only transfected Flag-REEP1 (calculated Mr = 23.4 kDa) (Lane 2); no endogenous REEP1 (calculated Mr = 22.3 kDa) expression was noted in untransfected cells (Lane 1). REEP1 mAb did not cross-react with Flag-REEP2 (Lane 3) or Flag-REEP6 (Lane 4). B. REEP2 polyclonal antisera identified both transfected Flag-REEP2 (calculated Mr = 29.4 kDa) (Lane 3) and endogenous REEP2 (calculated Mr = 28.3 kDa) in HEK293A cells (Lanes 1–4), but not Flag-REEP1 or Flag-REEP6. Note that both REEP1 and REEP2 demonstrate anomalous migration relative to their calculated molecular weights. C. Similar amounts of transfected Flag-REEP1, -REEP2, and –REEP6 were expressed, as detected by anti-Flag epitope mAb (M2). Molecular weight markers (kDa) are shown to right. Representative of three immunoblots and transfections.

Figure 2

Immunofluorescent characterization of REEP1 mAb HEK293A cells were transfected with either Flag-REEP1 or native, untagged-REEP1 cDNAs and analyzed by immunofluorescent wide-field microscopy with anti-Flag mAb (M2) and a new REEP1 mAb respectively. Cells were counterstained with either MitoTracker Red CMXROS or anti-calreticulin antisera, as described in Experimental Procedures. A. Immunofluorescent staining for Flag-REEP1 (M2 mAb) expressed in HEK293A cells revealed a fine reticular network that demonstrated a different localization pattern than mitochondria counterstained with MitoTracker Red CMXROS. B. REEP1 mAb detected a similar reticular staining pattern for untagged-REEP1, as seen above for Flag-REEP1. Mitochondria were counterstained with MitoTracker Red CMXROS. C. The fine reticular immunofluorescent pattern seen with M2 staining of Flag-REEP1 showed a similar localization as the ER marker protein calreticulin. D. REEP1 mAb delineated a similar reticular pattern for untagged-REEP1, when compared with anti-calreticulin staining. Bar = 10 μm. Representative of three transfections.

Figure 3

Expression of REEP1 and REEP2 proteins in various cell lines Left. Immunoblot analysis of REEP expression in commonly used cell lines. A. Representative immunoblot analysis of REEP1 expression using a new monoclonal antibody (NeuroMab Clone N345/51). No cell lines examined expressed endogenous REEP1. Immunoreactivity in Lane 6 represents transfected Flag-REEP1 (positive control). B. Representative immunoblot analysis of REEP2 expression in various cell lines using polyclonal REEP2 antisera. Lower molecular weight band correlated with endogenous REEP2, which was found in HEK293, HEK293A, and PC12 cells. Higher molecular weight band in Lane 7 represents transfected Flag-REEP2 (positive control). Larger immunoblots with loading controls are shown elsewhere (Supplemental Figure 1). Molecular weight markers (kDa) are shown to left. One hundred (Lanes 1–5) or forty (Lanes 6–7) micrograms of protein were loaded per lane. Representative of three immunoblots and transfections. Right: Analysis of REEP1 and REEP2 expression in NGF-treated PC12 cells. PC12 cells were treated with NGF for either 0 or 72 hours and immunoblot analysis was performed on whole cell lysates (75 microgram protein/lane). Flag-REEP1 transfected HEK29A or untransfected HEK293A membranes were also loaded as positive controls (50 microgram protein/lane). C. NGF-treatment did not induce REEP1 protein expression. D. REEP2 protein expression levels did not change following NGF treatment. Representative of three immunoblots and separate NGF treatments.

Figure 4

REEP1 and REEP2 protein expression in multiple tissues Representative immunoblot analysis of REEP1 and REEP2 expression in various tissues using REEP1 monoclonal antibody and REEP2 polyclonal antisera. A. REEP1 protein was detected only in brain, spinal cord, and testes. B. REEP2 protein was found in brain, spinal cord, adrenal gland, testes, and pituitary. Note absence of both REEP1 and REEP2 in astrocytes, skeletal muscle, heart, and other tissues. C. GAPDH was utilized as a positive loading control. Note differential expression seen amongst various tissues (Lanes 1–14), as described previously (Dittmer and Dittmer, 2006; Ferguson et al., 2005). Other loading controls were also examined (Supplemental Figure 3). Molecular weight markers (kDa) are shown to right. Representative of two immunoblots each.

Figure 5

REEP gene expression analysis in superior cervical and stellate sympathetic ganglia DNA microarray analysis was performed on total RNA isolated from superior cervical (SCG) and stellate (SG) sympathetic ganglia (n = 2, n = 3 respectively), to determine relative mRNA expression of various REEP family members. mRNA expression is represented on the y-axis in arbitrary fluorescence units (± SEM). In both SCG and SG, REEP1 and REEP5 demonstrated high expression, while REEP2 and REEP3 showed moderate expression levels. REEP4 and REEP6 were not detected (n.d.).

Figure 6

REEP1 expression in cultured SGN A. RT-PCR analysis of REEP1 expression in cultured SGN. RT-PCR was performed from total RNA isolated at various days of culture (n=3). Note that expression of REEP1 mRNA occurred between Day 4 and Day 8 of culture. GAPDH was utilized as a positive control for all reactions. B. Representative (n=3) immunofluorescent analysis of REEP1 expression in cultured SGN using REEP1 monoclonal antibody. Far Left. At Day 4 of culture, no REEP1 expression was seen in any cultured SGN examined. Calreticulin expression, an ER marker, was seen. Middle Left. At Day 8, extensive expression of REEP1 was noted in all cultured SGN examined. REEP1 was found in cell bodies, axons, and dendrites, revealing a similar localization pattern as the ER marker calreticulin. Middle Right. Similar extensive expression of REEP1 was seen in all cultured SGN examined at Day 16. Far Right. Note absence of REEP1, but expression of calreticulin, in non-SGN supporting cells Day 8. Non-SGN cells present include fibroblasts (arrow), satellite cells, and Schwann cells. Over one hundred cultured SGN were examined at each time point. Bar = 10 μm.