cell biology midterm but its me

EGFR

Epidermal Growth Factor Receptor; transmembrane protein involved with regulating cell growth, survival, and differentiation

What cancer cell line are we using and why

A431 cells are a human epidermoid carcinoma cell line known for overexpressing EGFR. they lack tumor suppressor protein p53

How does EGFR work

This binding triggers receptor dimerization and autophosphorylation of tyrosine residues in the intracellular domain, initiating a cascade of downstream signaling pathways

How are A431 cells cultured

under sterile conditions using Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serume (FBS) and 1% penicillin-streptomycin (Pen/Strep) to support growth and prevent contamination

What temperatures are A431 cells maintained in

37 degrees C in humidified incubator with 5% CO2

How are A431 cells harvested

Using RPPA Lysis Buffer

Protein quantification

Measuring protein concentration in cell lysates

SDS-PAGE

separating proteins by molecular weight

Western blotting

detecting specific proteins using antibodies

Immunofluorescence

Visualizing protein localization in cells.

Cell Fixation

Preserving cellular structures for analysis.

Protein synthesis

gene transcription, protein translation, post-translational modification

Transcription

DNA transcribed into mRNA in the nucleus. Travels to cytoplasm where ribosomes translate sequence into amino acids

What do amino acids form from transcription

polypeptide

What does the polypeptide chain undergo to become a functional protein

folding and modification

Each protein is made from a unique sequence of

20 different amino acids

What are amino acids linked by

peptide bonds

Primary structure

determines proteins final shape and function–linear amino acid sequence

Secondary structure

regular folding patterns like α helices and β sheets, formed

by hydrogen bonds in the backbone

Tertiary structure

the overall 3D shape of a single polypeptide, including loops and Folds.

Quaternary structure

the arrangement of multiple polypeptide chains (subunits) in a protein complex

What forces hold proteins

noncovalent interactions– hydrogen bonds, electrostatic attractions, van der waals forces, and hydrophobic interactions

Proteins have domains which are

compact, independently folding units within a polypeptide chain that serve specific structural or functional roles

Two examples of protein structures

collagen and keratin (also remember complex assemblies)

Chaperone proteins

prevent misfolding and aggregation

Amyloid structures

misfolded proteins may cause these which can cause alzheimers or parkisons

All proteins bind to other molecules called

ligands–through a binding site

What are the protein interactions stabilized by

noncovalent forces including hydrogen bonds, van der walls, electrostatic interactions, hydrophobic effects

Proteins undergo

conformational changes upon ligand binding which enhances or inhibits

Proteins can be regulated by

covalent modifications, such as phosphorylation, acetylation, or ubiquitination

The protein modifications can form

protein complexes or scaffolded assemblies that coordinate cellular functions

Cofactors/coenzymes

some proteins use to perform special tasks (like metal ions or small organic molecules)

Proteins can assemble into

larger structural complexes such as filaments, sheets, and spherical shells

Proteins are reinforced by

disulfide bonds—covalent linkages between cysteine residues

The cell membrane is

dynamic; composed of lipid bilayer with proteins

Lipid bilayer is

amphipathic hydrophilic heads and hydrophobic tails

Transmembrane proteins

span the bilayer and are amphipathic, with hydrophobic regions embedded in the membrane and hydrophilic regions exposed to the aqueous surroundings.

Monolayer-associated proteins

attached to the cytosolic side via an amphipathic α helix.

Lipid-linked proteins are

anchored to the membrane by covalently attached lipid groups

Peripheral proteins

bind indirectly through interactions with other membrane proteins and can be removed without disrupting the bilayer

Transmembrane proteins cross bilayer because

a helices allow hydrophilic backbone to form internal hydrogen bonds

Some channels are formed using

multiple a helices or B-barrel structures like porins

Glycocalyx

outer surface of plasma membrane coated with carbohydrates attached to lipids and proteins

What does glycocalyx do

protects cell, helps cell-cell recognition, and adhesion

How does EGF bind to EGFR

EGFR binds to ligand and has conformational changes that exposes dimerization motif. Allows two receptor molecules to form asymmetric dimers. This activates receptors kinese domain. Leads to trans-autophosphorylation of tyrosine residues.

Cell signaling allows for

cellular communication and allows cells to respond to env

Signal transduction

the conversion of one type of signal into another—is central to this process

Endocrine signaling

involves hormones traveling through the bloodstream to reach distant cells. For example, insulin from the pancreas regulates glucose uptake throughout the body.

Paracrine signaling

acts locally, with signal molecules diffusing through extracellular fluid to nearby cells This is common in wound healing and inflammation.

Autocrine signaling

occurs when a cell responds to signals it secretes itself, often seen in cancer cells promoting their own survival.

Neuronal signaling

uses electrical impulses and neurotransmitters to deliver rapid, targeted messages across synapses.

Contact-dependent signaling

requires direct membrane-to-membrane contact, crucial during development when adjacent cells influence each other’s fate

Signal transduction and receptor function

so extracellular molecule binds to a specific receptor either on the cell surface (hydrophilic or large) or inside (hydrophobic or small). then activates and trigger a transduction cascade. gene expression, metabolism, shape, or movement

Specificity and Diversity of Cellular Responses

cells can interpret signals differently depending on receptors

Intracellular Signaling Pathway

Once a receptor is activated, it triggers a cascade of intracellular signaling events. These pathways can

Phosphorylation-based switches

where protein kinases add phosphate groups to activate proteins, and , them to deactivate.phosphatases remove

GTP-binding proteins, which are active when bound to GTP and inactive when bound to GDP. These include trimeric G proteins (used by GPCRs) and monomeric GTPases like Ras

active with gtp inactive with gdp

EGFR relies on

phosphorylation-based switches (initial activation driven by tyrosine phosphorylation)

Antibodies enable

the detection, quantification, and localization of signaling molecules

Antibodies are immunoglobulin proteins naturally produced by B cells in response to antigens—foreign substances such as pathogens or toxins.

The anti-EGFR XP Rabbit antibody is used to

detect total EGFR protein levels as it’s activated

to further characterize the signaling cascade, we also utilize a

phospho-AKT antibody (used with western blotting)