Revisiting RAMP1 expression: Characterisation of antibodies against receptor activity-modifying protein 1 (RAMP1)

Abstract text (incl. figure legends and references)

Objective: Calcitonin gene-related peptide (CGRP) is well-established as a key component of migraine pathophysiology, yielding effective migraine therapeutics. Further refinement of these treatments requires a more in-depth understanding of CGRP's actions at its receptors, which contain a core accessory protein subunit: receptor activity-modifying protein 1 (RAMP1). Our understanding of RAMP1 expression at the cellular level is incomplete, partly due to the challenges in the availability and identification of specific and validated antibody tools.

Methods: We profiled antibodies for immunodetection of RAMP1 using western blotting, immunocytochemistry, and immunohistochemistry, including using RAMP1 knockout mouse tissue.

Results: Most antibodies could detect RAMP1 protein in western blotting and immunocytochemistry using transfected cells. Selected antibodies were profiled further, and two antibodies (844, ab256575) could detect a RAMP1-like band in western blots of rodent brain but not RAMP1 knockout mice. However, cross-reactivity with other proteins was evident for all antibodies. In immunohistochemistry clear conclusions about RAMP1 anatomical localization were prevented because each antibody unexpectedly detected distinct patterns of immunoreactivity.

Conclusions: Although most antibodies were capable of detecting RAMP1, their ability to also detect off-target proteins means that immunoreactivity produced by RAMP1 antibodies (including 844) in nervous tissue cannot be confidently attributed to RAMP1 in immunohistochemical applications. RAMP1 expression in nervous tissue therefore needs to be revisited. Our results have implications for using RAMP1 antibodies for any immunohistochemical investigation in any tissue. Furthermore, as RAMP1 is important for other GPCR/ligand pairings, our results have broader significance beyond the CGRP field.