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Underscoring the importance that direct cholesterol engagement has in the modulation of GLP-1R function, the changes in cholesterol binding and plasma membrane behaviours introduced by the V229A substitution led to significant effects on GLP-1R trafficking and signalling in beta cells: V229A caused a decrease in GLP-1R recruitment to CCPs and receptor internalisation, accompanied by an increase in G s (measured with mini-G s ) and -arrestin 2 engagement, as well as increased plasma membrane recycling following exendin-4 stimulation