Interaction of receptor-activity-modifying protein1 with tubulin

• Published in: Biochimica et biophysica acta Vol. 1770; no. 8; pp. 1145 - 1150
• Main Authors: Kunz, Thomas H., Mueller-Steiner, Sarah, Schwerdtfeger, Kerstin, Kleinert, Peter, Troxler, Heinz, Kelm, Jens M., Ittner, Lars M., Fischer, Jan A., Born, Walter
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.08.2007
• Subjects: Antigens, Viral, Tumor - physiology; Axons - metabolism; Calcitonin gene-related peptide; Calcitonin receptor-like receptor; Cell Line, Transformed; Cell Line, Tumor; Cell Transformation, Viral; Cloning, Molecular; Escherichia coli - genetics; Glutathione Transferase - metabolism; Humans; Intracellular Signaling Peptides and Proteins - chemistry; Intracellular Signaling Peptides and Proteins - genetics; Intracellular Signaling Peptides and Proteins - metabolism; Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS); Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Microscopy, Confocal; Neuroblastoma - pathology; Neurons - metabolism; Precipitin Tests; Protein interaction; Protein Structure, Tertiary; Proteins - metabolism; Receptor Activity-Modifying Protein 1; Receptor Activity-Modifying Proteins; Receptor-activity-modifying protein; Recombinant Fusion Proteins - metabolism; Simian virus 40 - physiology; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tubulin - chemistry; Tubulin - metabolism; Two-Hybrid System Techniques; β-tubulin; CTR; MALDI-TOF-MS; SDS; DTT; Receptor-activity-modifying protein; CLR; GST; PAGE; AM; β-tubulin; Calcitonin gene-related peptide; DMEM; RAMP; Protein interaction; CGRP; Calcitonin receptor-like receptor; Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS)
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2007.04.002

Receptor-activity-modifying protein (RAMP) 1 is an accessory protein of the G protein-coupled calcitonin receptor-like receptor (CLR). The CLR/RAMP1 heterodimer defines a receptor for the potent vasodilatory calcitonin gene-related peptide. A wider tissue distribution of RAMP1, as compared to that of the CLR, is consistent with additional biological functions. Here, glutathione S-transferase (GST) pull-down, coimmunoprecipitation and yeast two-hybrid experiments identified β-tubulin as a novel RAMP1-interacting protein. GST pull-down experiments indicated interactions between the N- and C-terminal domains of RAMP1 and β-tubulin. Yeast two-hybrid experiments confirmed the interaction between the N-terminal region of RAMP1 and β-tubulin. Interestingly, α-tubulin was co-extracted with β-tubulin in pull-down experiments and immunoprecipitation of RAMP1 coprecipitated α- and β-tubulin. Confocal microscopy indicated colocalization of RAMP1 and tubulin predominantly in axon-like processes of neuronal differentiated human SH-SY5Y neuroblastoma cells. In conclusion, the findings point to biological roles of RAMP1 beyond its established interaction with G protein-coupled receptors.