RESUMEN
Sturge-Weber syndrome (SWS) is a sporadic, congenital, neuro-cutaneous disorder characterized by a mosaic, capillary malformation. SWS and non-syndromic capillary malformations are both caused by a somatic activating mutation in GNAQ encoding the G protein subunit alpha-q protein. The missense mutation R183Q is the sole GNAQ mutation identified thus far in 90% of SWS-associated or isolated capillary malformations. In this study, we sequenced skin biopsies of capillary malformations from 9 patients. We identified the R183Q mutation in nearly all samples, but one sample exhibited a Q209R mutation. This new mutation occurs at the same residue as the constitutively-activating Q209L mutation, commonly seen in tumors. However, Q209R is a rare variant in this gene. To compare the effect of the Q209R mutation on downstream signaling, we performed reporter assays with a GNAQ-responsive reporter co-transfected with either GNAQ WT, R183Q, Q209L, Q209R, or C9X (representing a null allele). Q209L showed the highest reporter activation, with R183Q and Q209R showing significantly lower activation. To determine whether these mutations had similar or different downstream consequences we performed RNA-seq analysis in microvascular endothelial cells (HMEC-1) electroporated with the same GNAQ variants. The R183 and Q209 missense variants caused extensive dysregulation of a broad range of transcripts compared to the WT or null allele, confirming that these are all activating mutations. However, the missense variants exhibited very few differentially expressed genes (DEGs) when compared to each other. These data suggest that these activating GNAQ mutations differ in magnitude of activation but have similar downstream effects.
Asunto(s)
Síndrome de Sturge-Weber , Capilares/anomalías , Células Endoteliales/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Humanos , Mutación/genética , Subunidades de Proteína/metabolismo , Síndrome de Sturge-Weber/genética , Síndrome de Sturge-Weber/metabolismo , Síndrome de Sturge-Weber/patología , Malformaciones VascularesRESUMEN
Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) are potent vasodilator peptides and serve as ligands for the G-protein coupled receptor (GPCR) calcitonin receptor-like receptor (CLR/Calcrl). Three GPCR accessory proteins called receptor activity-modifying proteins (RAMPs) modify the ligand binding affinity of the receptor such that the CLR/RAMP1 heterodimer preferably binds CGRP, while CLR/RAMP2 and CLR/RAMP3 have a stronger affinity for AM. Here we determine the contribution of each of the three RAMPs to blood pressure control in response to exogenous AM and CGRP by measuring the blood pressure of mice with genetic reduction or deletion of the receptor components. Thus, the cardiovascular response of Ramp1-/-, Ramp2+/-, Ramp3-/-, Ramp1-/-/Ramp3-/- double-knockout (dKO), and Calcrl+/- mice to AM and CGRP were compared to wildtype mice. While under anesthesia, Ramp1-/- male mice had significantly higher basal blood pressure than wildtype males; a difference which was not present in female mice. Additionally, anesthetized Ramp1-/-, Ramp3-/-, and Calcrl+/- male mice exhibited significantly higher basal blood pressure than females of the same genotype. The hypotensive response to intravenously injected AM was greatly attenuated in Ramp1-/- mice, and to a lesser extent in Ramp3-/- and Calcrl+/- mice. However, Ramp1-/-/Ramp3-/- dKO mice retained some hypotensive response to AM. These results suggest that the hypotensive effect of AM is primarily mediated through the CLR/RAMP1 heterodimer, but that AM signaling via CLR/RAMP2 and CLR/RAMP3 also contributes to some hypotensive action. On the other hand, CGRP's hypotensive activity seems to be predominantly through the CLR/RAMP1 heterodimer. With this knowledge, therapeutic AM or CGRP peptides could be designed to cause less hypotension while maintaining canonical receptor-RAMP mediated signaling.