It is well established that protein interactions with phospholipids, particularly phosphoinositides, serve to regulate many different cellular processes. Due to the charged nature of biomembranes, electrostatic interactions are particularly effective between proteins and lipids at membrane interfaces where protein oligomerization and locally high charge densities of certain phospholipids can contribute significantly to cellular processes. Phosphoinositide distributions contribute to organelle identity, recruit proteins important for anterograde or retrograde trafficking to the right place at the right time, and directly influence the capacity of proteins to signal to downstream partners. Our studies provide evidence in support of the importance of protein-lipid interactions in the regulation of epidermal growth factor receptor (EGFR) activity and signaling, as well as in biosynthetic trafficking from the endoplasmic recticulum (ER) to the plasma membrane (PM). The polybasic juxtamembrane (JX) region of EGFR has been proposed to regulate protein activity by interacting with the PM and thus preventing tyrosine kinase domain activation. We demonstrate that reduction of the net charge of the JX region in wild-type EGFR results in constitutive activation of the receptor and conferral of its capacity to transform cells in a ligand-independent manner. These capabilities are maintained when the receptor is retained in the endoplasmic reticulum. In addition, we show that the polybasic JX region also plays a positive role in the response of EGFR to ligand. Receptors in which the net positive charge of the JX region is reduced show reduced activation and are deficient in downstream signaling, including their capacity to mediate Ca2+ mobilization. In addition, we demonstrate that protein-lipid interactions also contribute to proper biosynthetic trafficking in cells. We provide pharmacological and molecular genetic evidence for the functional requirement of a pool of phosphoinositide 4-phosphate, synthesized by phosphoinositide 4-kinase III[alpha], for ER to PM protein trafficking. In summary, strategies to perturb both protein structure and phospholipid availability provide evidence in support of roles for basic amino acid sequences and negatively charged phospholipids in biosynthetic trafficking and cellular signaling regulation.